Phase I/II Study of PROSTVAC in Combination With Nivolumab in Men With Prostate Cancer
Phase: PHASE1
Conditions: Prostate Cancer
Sponsor: National Cancer Institute (NCI)



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PRÉCIS 
Background:

Immune checkpoint inhibitors interfere with the immune system’s autoregulatory 
mechanisms, allowing for a potentially expanded and prolonged T-cell response with the 
possibility of greater antitumor effects.

Nivolumab is a fully human IgG4 monoclonal antibody that targets the PD-1 protein.  
Specifically, the antibody binds to the PD-1 receptor and blocks its interaction with PD-
L1 and PD-L2, thereby releasing PD-1 pathway-mediated inhibition of the immune 
response, including anti-tumor immune response. 

PROSTVAC (developed by the National Cancer Institute [NCI] and licensed to Bavarian 
Nordic Immunotherapeutics, Mountain View, CA) is a therapeutic cancer vaccine for 
prostate cancer. Early studies have demonstrated immunologic efficacy and suggested 
clinical benefit.  A phase III trial has completed accrual.

A previous study combining the immune checkpoint inhibitor ipilimumab and 
PROSTVAC suggested greater efficacy than PROSTVAC alone.  Additional studies have 
demonstrated the potential efficacy of immunologic combination therapy with the 
immune checkpoint inhibitor nivolumab.  

This study will aim to evaluate the impact of the combination of PROSTVAC and the 
immune checkpoint inhibitor nivolumab on the tumor microenvironment focusing on 
immune cell infiltration as the primary endpoint.  

US-MRI imaging technology will be employed to sample the tumor before treatment and 
after radical prostatectomy.  

The findings from this study could serve as the basis for future studies with this 
combination in this population of participants and more advanced disease.
Objectives:

Safety (For castration resistant prostate cancer (CRPC) lead-in cohort)

Evaluate changes in T-cell infiltration in the tumor after neoadjuvant treatment with 
PROSTVAC and nivolumab, relative to changes seen in a phase 2 trial with PROSTVAC 
alone in the neoadjuvant setting- NCT02153918 (For the neoadjuvant cohort). 
Eligibility:

Participants must have histopathological documentation of adenocarcinoma of the 
prostate prior to starting this study and evaluable biopsy tissue (e.g., unstained slides or 
blocks) available for analysis. 

For the castration resistant lead in cohort, if histopathological documentation is 
unavailable, a rising PSA and a clinical course consistent with prostate cancer would be 
acceptable.

Participants must have a performance status of 0 to 1 according to the ECOG criteria.

Hematological eligibility parameters (within 16 days of starting therapy):

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o
Granulocyte count 1,500/mm3
o
Platelet count 100,000/mm3
o
Hgb ≥ 8 g/dL 
o
Biochemical eligibility parameters (within 16 days of starting therapy):
o
Hepatic function: Bilirubin < 1.5 mg/dl (OR in participants with Gilbert’s syndrome, total 
bilirubin ≤ 3.0 mg/dL), AST and ALT ≤ 2.5 times upper limit of normal.
o
Creatinine ≤ 1.5 X ULN
Design:

The primary focus of this study will be to evaluate PROSTVAC and nivolumab in the 
neoadjuvant setting. 

Lead-in cohort evaluating the safety and tolerability of this combination in the castration 
resistant setting (CRPC cohort)

Following this lead-in cohort in the CRPC setting, we will enroll a cohort in the 
neoadjuvant setting evaluating the combination of PROSTVAC and nivolumab. 

The lead-in safety cohort will require 10 participants and the neoadjuvant cohort will 
require 17 evaluable participants. In order to allow for a small number of inevaluable 
participants, the accrual ceiling will be set to 29 participants.

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TABLE OF CONTENTS 
PRÉCIS...........................................................................................................................................2
TABLE OF CONTENTS ................................................................................................................4
STATEMENT OF COMPLIANCE ................................................................................................7
1
INTRODUCTION ...................................................................................................................7
1.1
Study Objectives ...............................................................................................................7
1.2
Background and Rationale................................................................................................8
2
ELIGIBILITY ASSESSMENT AND ENROLLMENT .......................................................18
2.1
Eligibility Criteria ...........................................................................................................18
2.2
Participant Registration and Status Update Procedures..................................................22
2.3
Baseline Evaluation.........................................................................................................23
3
STUDY IMPLEMENTATION .............................................................................................24
3.1
Study Design...................................................................................................................24
3.2
Drug Administration .......................................................................................................25
3.3
Dose Modifications.........................................................................................................26
3.4
Stopping Rules ................................................................................................................26
3.5
Study Calendar................................................................................................................28
3.6
Cost and Compensation ..................................................................................................34
3.7
Criteria for Removal from Protocol Therapy and Off Study Criteria.............................34
3.8
Follow-up Evaluations ....................................................................................................35
3.9
Post-Study Evaluation.....................................................................................................35
4
CONCOMITANT MEDICATIONS/MEASURES...............................................................35
5
CORRELATIVE STUDIES FOR RESEARCH ...................................................................36
5.1
Immune cell infiltrate (Tissue Specimens) .....................................................................36
5.2
Sample Storage, Tracking and Disposition.....................................................................44
6
DATA COLLECTION AND EVALUATION .....................................................................46
6.1
Data Collection ...............................................................................................................46
6.2
Data Sharing Plans..........................................................................................................47
6.3
Response Criteria (Applicable to CRPC cohort) ............................................................48
6.4
Toxicity Criteria..............................................................................................................55
7
NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN.....55

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7.1
Definitions.......................................................................................................................55
7.2
OHSRP Office of Compliance and Training/  IRB  Reporting.......................................55
7.3
NCI Clinical Director Reporting.....................................................................................56
7.4
Institutional Biosafety Committee (IBC) Reporting Criteria..........................................56
7.5
NIH Required Data and Safety Monitoring Plan............................................................57
8
SPONSOR PROTOCOL/SAFETY REPORTING................................................................58
8.1
Definitions.......................................................................................................................58
8.2
Assessment of Safety Events ..........................................................................................59
8.3
Reporting of Serious Adverse Events .............................................................................59
8.4
Safety Reporting Criteria to the Pharmaceutical Collaborators......................................60
8.5
Reporting Pregnancy.......................................................................................................61
8.6
Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND...................62
8.7
Sponsor Protocol Deviation Reporting ...........................................................................62
9
CLINICAL MONITORING..................................................................................................62
10
STATISTICAL CONSIDERATIONS ..................................................................................63
11
COLLABORATIVE AGREEMENTS..................................................................................65
11.1
Collaborative Research and Development Agreement (CRADA)..............................65
11.2
Clinical Trials Agreement (CTA)................................................................................65
11.3
Materials Transfer Agreement (MTA)........................................................................65
12
HUMAN SUBJECTS PROTECTIONS................................................................................65
12.1
Rationale For Subject Selection ..................................................................................65
12.2
Inclusion of Women and Minorities............................................................................65
12.3
Participation of Children .............................................................................................65
12.4
Participation of Subjects Unable to Give Consent ......................................................65
12.5
Evaluation of Benefits and Risks/Discomforts ...........................................................66
12.6
Risks/Benefits Analysis...............................................................................................67
12.7
Consent Process and Documentation ..........................................................................68
13
REGULATORY AND OPERATIONAL CONSIDERATIONS..........................................69
13.1
Study Discontinuation and Closure.............................................................................69
13.2
Quality Assurance and Quality Control ......................................................................69
13.3
Conflict of Interest Policy ...........................................................................................70
13.4
Confidentiality and Privacy.........................................................................................70

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PHARMACEUTICAL INFORMATION .............................................................................71
14.1
Recombinant Fowlpox-PSA(L155)TRICOM..........................................................71
14.2
Recombinant Vaccinia-PSA(L155)/TRICOM........................................................73
14.3
Nivolumab...................................................................................................................83
15
REFERENCES ......................................................................................................................86
16
APPENDICES .......................................................................................................................91
16.1
Appendix A: Performance Status Criteria...................................................................91
16.2
Appendix B: Vaccinia-PROSTVAC Patient Instruction Sheet...................................92
16.3
Appendix C: Management Algorithms For Endocrinopathy, Gastrointestinal, Hepatic, 
Neurological, Pulmonary, Renal, And Skin Adverse Events ....................................................95
16.4
Appendix D: Immune Related Response Criteria .....................................................102

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STATEMENT OF COMPLIANCE
The trial will be carried out in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and the following: 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812) 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training.
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; an 
IRB determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form.
1
INTRODUCTION
1.1
STUDY OBJECTIVES
1.1.1
Primary Objective

Safety (For castration resistant prostate cancer (CRPC) lead-in cohort)

Evaluate changes in T-cell infiltration in the tumor after neoadjuvant treatment with 
PROSTVAC and nivolumab, relative to changes seen in a phase 2 trial with PROSTVAC 
alone in the neoadjuvant setting – NCT02153918 (For neoadjuvant cohort). 
1.1.2
Secondary Objectives

To determine the change in peripheral PSA-specific T cells in participants treated with 
PROSTVAC and nivolumab.

To document any intraprostatic Treg cell infiltration with CD4+FOX-P3 staining.

To document any PSA changes secondary to immune treatment, including rate of 
biochemical recurrence after prostatectomy.

To document any MRI changes secondary to treatment (including changes in ADC 
mapping)

To evaluate changes in PDL-1 expression in tumor.

To document pathologic responses (including pathologic CR).

To evaluate changes in immune cell subsets in the periphery.

To evaluate changes in soluble immune mediating factors (such as cytokines, etc.) in 
sera.

To evaluate changes in circulating tumor cells levels (for CRPC cohort only)

Safety (for localized prostate cancer cohort)

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1.2
BACKGROUND AND RATIONALE
It is expected that 26,120 men will die from prostate cancer in the US in 2016.[1-3] While some 
men are definitively treated for localized disease, many men will ultimately develop castrate 
resistant prostate cancer (CRPC) and succumb to the disease; 2 this is due to the lack of an 
optimal treatment strategy. Treatment options typically involve radical prostatectomy (RP) or 
radiation therapy (RT) in combination with androgen deprivation therapy (ADT). Following RP 
upwards of 50% of patients with high-risk disease will experience a biochemical recurrence at 5 
years, [4] and approximately 20% will die of their disease in 10-15 years. [5]  
1.2.1
Immunotherapy in prostate cancer
Vesalainen et al [6] found that the density of tumor-infiltrating lymphocytes in primary prostatic 
adenocarcinoma was independent of the tumor differentiation (Gleason score), and low numbers 
of tumor-infiltrating lymphocytes were an independently negative predictor of survival (n=325, 
P<0.05) suggesting a role the immune system may have in the control of this disease.
Several characteristics of prostate cancer make it an ideal target for immunotherapy.  Its 
relatively indolent disease course allows sufficient time to generate immune responses, which 
usually take weeks or months to mount. Therapeutic vaccines for prostate cancer offer a 
therapeutic approach and recent data have renewed interest in this form of treatment for 
metastatic prostate cancer.[7]  
The goal of therapeutic cancer vaccines is to generate a targeted immune response leading to 
immune-mediated anti-tumor activity.  Sipuleucel-T is a therapeutic cancer vaccine generated 
from peripheral blood mononuclear cells obtained from individual patients via leukapheresis. 
This vaccine is generated after a patient’s peripheral immune cells are collected via 
leukapheresis, transported to a regional processing center where they are exposed in vitro to a 
PAP/GM-CSF fusion protein. At the end of this process, the activated cellular product is re-
infused into the patient. A full course of therapy repeats this process 3 times every 2 weeks for 1 
month [8, 9].  A phase III trial (n = 512) demonstrated an overall survival benefit for the vaccine 
(25.8 months vs. 21.7 months; P = 0.032)[10]. Based on these overall survival findings, the FDA 
approved sipuleucel-T for the treatment of asymptomatic or minimally symptomatic mCRPC, 
making it the first FDA-approved therapeutic cancer vaccine for the treatment of any 
malignancy. 
1.2.2
PROSTVAC
PROSTVAC, an off-the-shelf therapeutic cancer vaccine, offers an alternative strategy to 
enhance immune targeting of the tumor antigen, PSA.[11] PROSTVAC employs genetically 
altered poxviruses to deliver targeting information to immune cells and generate an immune 
response. Administered subcutaneously, the poxviruses deliver the transgenes for the tumor 
associated antigens (PSA) to cells including antigen presenting cells through cellular infection. 
Once these pox viruses are within the cellular cytoplasm, the transgenes are processed. The end 
result is an antigen presenting cell expressing a PSA peptide within the major histocompatibility 
complex, resulting in PSA-specific cytotoxic T lymphocytes activation.[11] (Figure 1) This 
approach does not require logistically complex, labor-intensive ex vivo preparation of patients’ 
peripheral blood. PROSTVAC is thus potentially more feasible and practical over the long-term 
than sipuleucel-T[12].

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Early clinical trials demonstrated that PROSTVAC was well tolerated with common side effects 
including injection site reactions and flu-like symptoms.[13] PROSTVAC has subsequently been 
investigated in 2 phase II trials in metastatic CRPC (mCRPC), both of which administered the 
vaccine at monthly intervals until disease progression. An industry-sponsored, placebo-
controlled, multicenter trial in 125 mCRPC patients randomized them 2:1 in favor of 
PROSTVAC; the placebo was an empty poxviral vector containing no transgenes. As was seen 
in the sipuleucel-T studies, patients receiving vaccine showed no change in progression free 
survival, yet had an overall survival benefit (25.1 months with PROSTVAC  vs. 16.6 months 
with placebo; P = 0.0061).[14]  (Figure 2) A second phase II study of PROSTVAC of 32 
mCRPC patients at the NCI demonstrated that the vaccine was able to generate a T-cell specific 
immune response and patients with the greatest magnitude of this response appeared to have 
superior outcomes. [15]  
The immune impact induced by PROSTVAC has also been described more broadly.  Of 104 
patients tested for T-cell responses, 57% (59/104) demonstrated a ≥2-fold increase in PSA-
specific T cells 4 weeks after vaccine (median 5-fold increase) compared with pre-vaccine.  In 
addition, 68% (19/28) of patients tested mounted post-vaccine immune responses to tumor-
associated antigens not present in the vaccine demonstrating the ability of the immune response 
to expand once generated in vivo (antigen spreading).[16]  Based on the findings in these early 
trials, a phase III trial of PROSTVAC in mCRPC is currently underway (NCT01322490).
Figure 1. Poxviral vaccine strategy: Modified 
poxvirus contains transgenes for the tumor-associated 
antigen PSA and 3 T-cell costimulatory molecules 
[11]                                

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Figure 2. PROSTVAC improved survival in 
mCRPC patients in a randomized multicenter phase II 
trial.[14]
1.2.3
Immune Checkpoint Inhibitors 
1.2.3.1 Ipilimumab
Immune checkpoint inhibitors interfere with the immune system’s autoregulatory mechanisms, 
allowing for a potentially expanded and prolonged T-cell response with the possibility of greater 
antitumor effects. [17]
Ipilimumab, a fully human anti-CTLA-4 monoclonal antibody, is a first-in-class immune 
checkpoint inhibitor. CTLA-4 is expressed on cytotoxic T lymphocytes (CTLs) after activation 
by APCs. The CTLA-4 receptor on CTLs is a negative regulator of T-cell activation that 
outcompetes CD28 for binding to B7 on APCs. In contrast to CD28/B7 binding, which acts as a 
costimulatory signal, the binding of CTLA-4 by ipilimumab removes the physiologic brake, 
augmenting the immune response by blocking the interaction of CTLA and B7.[17] Preclinical 
and clinical studies also suggest ipilimumab may also increase T-cell avidity, decrease Tregs 
within tumor), and expand the T-cell repertoire. [18-20] 
Ipilimumab was FDA-approved in 2011 for the treatment of unresectable or metastatic 
melanoma. The approval was based on a randomized (3:1:1) double-blind clinical trial that 
demonstrated an OS benefit in patients receiving ipilimumab; but, similar to the earlier trials 
involving sipuleucel-T and PROSTVAC, there was no short term change in disease 
progression.[21]  Despite the clinical efficacy, because ipilimumab generates a more non-
specific immune response, it characteristically has frequent immune related adverse events 
(irAEs).  IrAEs are mechanism-based adverse events and chiefly include skin and mucosal rash, 
diarrhea/colitis, hepatotoxicity, and hypophysitis.[21] Immunosuppressive agents such as 
systemic steroids are the standard treatment for many irAEs which are thus reversible.  For irAEs 
that impact endocrine glands, replacement hormones may be required.
Ipilimumab has also been investigated in mCRPC.  A recently completed phase III trial 
compared ipilimumab and limited radiation (serving as an immune enhancing therapy) to limited 
radiation and placebo.  This study was conducted in advanced mCRPC, patients who had 
progressive disease on docetaxel and thus had only a limited expected survival (approximately 1 
year).  The study did not meet its primary endpoint of overall survival, but results of this trial 
showed a median overall survival favoring ipilimumab over placebo (11.2 vs. 10 months; HR 
0.85, 95% CI 0.72–1.00; p = 0.053.[22]  Interestingly PSA declines of ≥ 50% in evaluable 

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patients (13.1% vs. 5.3%) favored patients getting ipilimumab. In subset analyses, patients with 
more indolent disease features had a substantially improved survival if they were treated with 
ipilimumab compared to placebo (22.7 vs. 15.8 months; n=142; HR 0·62, 95% CI 0·45–0·86; 
p=0·0038). These results support the concept that immunotherapy given earlier in the course of 
disease may yield greater benefit. [23]  An on-going second phase III trial of ipilimumab vs. 
placebo in chemotherapy-naïve mCRPC patients has completed accrual and results are 
anticipated in the next year (NCT01057810). Given that these patients have an earlier stage of 
disease, the results from this study will suggest whether this is indeed a more appropriate 
population of patients.
1.2.4
Anti-PD1 and Anti-PDL1
Agents blocking programmed cell death protein-1 (PD-1) and its ligand PD-ligand-1 (PD-L-1) 
are other examples of immune checkpoint molecules shown to have antitumor activity in solid 
malignancies. [24, 25]  These interventions represent a form of immune checkpoint inhibition 
disrupting the interaction between PD1 (e.g., on immune cells) and PDL1 (e.g. on tumor 
cells).[26] 
1.2.4.1 Nivolumab
Nivolumab is a fully human IgG4 monoclonal antibody that targets the PD-1 protein.  
Specifically, the antibody binds to the PD-1 receptor and blocks its interaction with PD-L1 and 
PD-L2, thereby releasing PD-1 pathway-mediated inhibition of the immune response, including 
anti-tumor immune response. 
Nivolumab was granted accelerated approval by the US FDA in December of 2014 for the 
treatment of patients with unresectable or metastatic melanoma and disease progression 
following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. Approval was 
based on objective response rate (ORR) and durability of response in the first 120 patients who 
were treated with nivolumab and had a minimum 6 months follow up from an ongoing, 
randomized, open-label trial in which 370 patients with unresectable or metastatic melanoma 
received nivolumab 3 mg/kg intravenously every 2 weeks (n=268) or investigators choice of 
chemotherapy (n=102).  Patients were excluded from the trial if they had an autoimmune disease, 
a medical condition that required corticosteroids or immunosuppression, or a history of severe 
ipilimumab-related adverse reactions. 
The major efficacy endpoints were confirmed ORR and response duration. The ORR was 32% 
(95% CI:23,41) with four complete responses and 34 partial responses. At the time of the FDA 
approval, five responding patients had progressed, while the remaining 33 patients (87%) had 
ongoing responses (range 2.6+ to 10+months).  Thirteen patients had ongoing responses of 6 
months or longer. The most common adverse reactions noted were rash in greater or equal to 
20%. Clinically significant immune-mediated adverse reactions included pneumonitis, colitis, 
hepatitis, nephritis, hypo and hyperthyroidism.[27] 
As of August 2021, nivolumab is FDA approved for 10 different cancers, some with multiple 
lines of approval [28]. As with all inhibitors of PD1/PDL1 signaling, nivolumab can cause 
inflammation in virtually any organ or tissue in the body. Per the Nivolumab Investigator 
Brochure version 21 the side effects include: 

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Very Common Side Effects (Occurring in greater than 1 in 10 people)

Fatigue 

Diarrhea

Rash, itching 
Common Side Effects (Occurring in up to 1 in 10 people)

Nausea

Vomiting 

Constipation

Mouth ulcers and sores

Dry mouth

Decreased appetite

Fever

Chills (feeling cold)

Muscle pain, weakness, stiffness, spasms 

Joint pain or stiffness

Headache

Dizziness, fainting

Abdominal pain

Difficulty breathing

Cough

Dry skin

Skin color changes

Allergic reactions during/after infusion

Infusion related reactions

Anemia, may require blood transfusion

Thrombocytopenia

Pain or swelling in your arms, legs, ankles or body

Peripheral neuropathy 

Inflammation of the colon (colitis) 

Inflammation of the lung (pneumonitis) 

Abnormal blood chemistry, including abnormal liver, kidney, and pancreas function tests 

High blood sugar (hyperglycemia)

Hypothyroidism

Hyperthyroidism

Hyponatremia
Uncommon Side Effects (Occurring in up to 1 in 100 people)

Alopecia

Adrenal insufficiency

Hypertension 

Hypotension 

Fast heart rate

Irregular heart rhythm

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
Blurry vision, dry eyes, eye inflammation

Orbital myositis

Inflammation of the liver (hepatitis) – Can be fatal

Hives

Erythema multiforme

Psoriasis

Pemphigoid 

Can’t sleep

Neutropenia

Inflammation of the kidney, kidney failure requiring dialysis

Dehydration

Upper respiratory infections

Pancreatitis 

Inflammation of the pituitary gland

Condition in which the pituitary gland does not produce enough hormones

Sarcoidosis

Bronchitis

Inflammation of the mouth and lining of digestive tract

Inflammation of the muscles

Hypoxia

Diabetes mellitus 
Rare Side Effects (Occurring in up to 1 in 1000 people)

Increased blood creatine phosphokinase levels

Increased blood acid levels caused by diabetes

Blood glucose increased for too long causing severe dehydration and confusion caused by 
diabetes

Rosacea

Fluid around lungs

Inflammation of the brain (encephalitis) – Can be fatal

Inflammation of the blood vessels which could cut off blood supply to tissues and organs

Inflammation of the duodenum

Cranial nerve disorder which may affect smell, taste, vision, sensation in the face, facial 
expression, hearing, balance, speech, swallowing and muscles of the neck

Guillain-Barre Syndrome

Anaphylaxis

Drug hypersensitivity

Rhabdomyolysis and polymyositis 

Toxic epidermal necrolysis

Stevens-Johnson syndrome 

Myasthenia gravis

Polymyalgia rheumatica

Inflammation of the spinal cord

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
Inflammation of the heart (myocarditis)

Liver injury

Double vision

Demyelination or loss of protective coating around your nerve fibers

Meningitis or swelling of the brain or spinal cord membranes

A hole developing in your intestinal tract

Haemophagocytic lymphohistiocytosis

Kikuchi- Fujimoto disease
The Nivolumab investigator brochure may be referenced for additional information. 
While there doesn’t appear to be significant activity of anti-PD1 or anti-PDL1 targeted agents 
alone in prostate cancer outside of the ~5% of mCRPC that are MSI hi [29], it is likely because 
this is a T-cell poor tumor with little underlying immune response to unleash. [30] A vaccine 
could potentially turn this into a T-cell enriched tumor as described below.
1.2.5
Rationale to Combine Immunotherapies 
1.2.5.1 Immunogenic intensification 
One of the most compelling areas of study in immunotherapy is the effects of combining 
immunotherapeutic approaches, a process known as immunogenic intensification. Novel 
approaches such as combining vaccine with immune checkpoint inhibitors highlight the potential 
of immunogenic intensification, whereby combining immunotherapies could potentially generate 
a greater immune response and enhance tumor-cell killing. [31] Previous clinical studies have 
shown evidence of enhanced clinical outcomes with manageable toxicity. 
A phase I study treated 30 patients with mCRPC with a fixed dose of PROSTVAC in 
conjunction with escalating doses of ipilimumab given at monthly intervals. No dose-limiting 
toxicities were seen and again side effects typical of ipilimumab were observed.  Of the 24 
patients who were chemotherapy-naïve, 14 (58%) had PSA declines, six of which (25%) were > 
50%. The median OS of patients treated with this combination was 34.4 months.  This OS 
compares favorably to other studies with PROSTVAC and sipuleucel-T which employed vaccine 
alone in the same population with similar prognostic features. [32],[15],[14] 
As expected, the range of toxic effects identified exceeded those in single-agent studies 
involving PROSTVAC, where grade 1 or 2 local injection-site reactions were most common.  
However, the proportion of patients affected by grade 3-4 toxic effects in this study (eight [27%] 
of 30 patients) was similar to previous phase I trials involving ipilimumab. [33],[34],[12]  These 
data suggest that the combination of a vaccine that enhances co-stimulation with an immune 
checkpoint inhibitor does not seems to be associated with increased immune-related adverse 
events compared with ipilimumab alone. [23]
A second phase I study that combined the whole tumor cell vaccine GVAX with escalating doses 
of ipilimumab (0.3 – 5 mg/kg) in patients with mCRPC.[35] Seven patients (25%) who received 
either 3 or 5 mg/kg ipilimumab had PSA declines of ≥ 50% while 2 patients showed a clear 
regression of bone metastases.  irAEs were similar in incidence and character to those that had 
been previously observed with single agent ipilimumab. 

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These findings provide a clinical proof of concept for use of an immune checkpoint inhibitor in 
combination with a therapeutic cancer vaccine in the treatment of mCRPC.  Given the limited 
toxicity profile of anti-PDL1 treatments, there may be stronger rationale for the use PROSTVAC 
and antiPDL1 in patients with prostate cancer, although randomized trials are required to 
estimate any potential clinical benefit of such treatments.
1.2.5.2 Immune Response Can Drive PDL1 Expression in the Tumor
Preliminary data also suggests that an activated immune response, particularly those associated 
with increased INF-gamma in the tumor microenvironment can drive PDL1 expression on cancer 
cells, making it a relevant target for a PDL1 inhibitor.  Based on this rationale, not only would a 
therapeutic cancer vaccine potentially have a therapeutic anti-tumor impact, associated CD8 T-
cell activation and the ensuing production of INF-gamma could drive PDL1 expression on the 
tumor cells.  These data provide further rationale for the combination of a therapeutic cancer 
vaccine and anti-PDL1 therapy. [36]
Preclinical data with therapeutic vaccines suggests that one can give tumor associated antigen 
(TAA) based vaccines (where the TAA is a selfantigen and the tumor doesn’t express PDL1) and 
induce a response that leads to infiltration of activated T-cells and subsequent up-regulation of 
PDL1 expression (NCI preclinical data, not published).
1.2.6
Rationale for Evaluating Immunotherapies in the Neoadjuvant Setting 
The neoadjuvant setting of prostate cancer provides an opportunity to assess the immunologic 
impact of these therapies in the tumor microenvironment.  This proof of concept has been 
provided by a study of sipuleucel-T in untreated prostate cancer.  When given in the neoadjuvant 
setting prior to radical prostatectomy sipuleucel-t was associated with increased immune cells 
seen at the tumor/prostate interface (periphery of the tumor). [37]  A trial of PROSTVAC alone 
prior to radical prostatectomy (NCT02153918) has completed accrual and evaluation of the 
immunologic impact on the tumor is in progress. Data from that study will be used for 
comparison in this study.  
The data acquired from the tumor microenvironment in this study could provide the rationale for 
a larger clinical trial in this same population to determine if combination immunotherapy 
(checkpoint inhibitors and vaccine) could enhance cure rate or significantly impact time to 
disease recurrence.  In addition, data from this study could be extrapolated to more advanced 
disease and provide rational for combination immunotherapy in those patients as well.  The 
findings of this study could also contribute to biomarker development in the use of 
immunotherapy and immunotherapy combinations. 
1.2.6.1.1  Evaluation using multiparametric MRI imaging of the prostate
As described below, patients will have multiparametric MRI imaging before and after 
neoadjuvant immunotherapy.  This will provide an opportunity to evaluate qualitative and 
quantitative changes in the multiparametric MRI.  A previous study here at the NCI 
(NCT01496131) has demonstrated that immunotherapy combined with ADT led to substantial 
improvements in tumor density (i.e. decreased tumor density as measured by ADC mapping) in 
patients who received vaccine with ADT compared to ADT alone.  This will be prospectively 
evaluated in this trial. 

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1.2.7
MR-US Technology to Acquire and Compare Tissue prior to Treatment and after Surgery
The MR-US “fusion” approach pioneered by Drs. Pinto and Choyke can be used to specifically 
biopsy lesions seen on mpMRI.  Such targeted biopsies will be evaluated in the men prior to 
neoadjuvant combination immunotherapy. All men will have a 3T MR examination and MR/US 
fusion guided biopsy will utilize custom biopsy probe with the embedded passive 
electromagnetic (EM) tracker that allows real-time tracking of the US image and needle location. 
3D reconstructed US volume will be registered and fused with the pre-biopsy MRI dataset. 
Continuous correction of rigid motion between the reconstructed reference 3D US volume and 
real-time 2D US images will enable tracking of the target lesions over the course of the biopsy 
procedure. The technical aspects and validation of this approach have been described earlier.[38] 
Biopsy targets identified in the MRI and re-identified in TRUS by means of registration will be 
sampled under real-time US guidance. The EM tracking and fusion technology we plan to use for 
this procedure has been developed and thoroughly validated by the NCI and has been applied in 
more than 195 cases. [39, 40]  Data from these biopsies will establish baseline immunologic 
assessments of the tumor microenvironment.  
Custom made patient –specific MR based prostate gland molds: A 3D model of the prostate 
can be created in a novel technique developed and pioneered in the NIH Clinical Center (Figure 
3), as described previously.[41] In brief it is a 3D model based upon MR images, which begins 
with manual segmentation of the MR T2W images.  The prostate is outlined and converted via 
software into a 3D object. This is then imported into SolidWorks (SolidWorks, Dassault 
Systèmes SolidWorks Corp., Concord, MA, USA). There it is subtracted to create an internal 
cavity that precisely shapes the patients’ gland. Slots for slicing the gland at 6mm intervals are 
designed and orientation markings (Superior/Inferior, Right/Left etc.) are added and it is then 
created using a 3D printer
Figure 3: Customized, patient-specific prostate 
specimen mold. (a) Three-dimensional 
representation of final prostate mold in green, 
shown in two halves with a 3D model of the 
patient’s prostate shown in brown. (b) Mold is 
created using a 3D printer, shown here in two 
halves fit together using small pegs/slots in the 
corners.
The mold approach has also been shown to allow for decision system support development [42] 
that provides a probability map of peripheral zone cancers based upon MRI.  This expertise will 
be critical to the imaging, allowing suspicious lesions to be targeted for biopsy and subsequent 

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analyses (of immune cells in the tumor microenvironment after neoadjuvant therapy) to be 
performed on the tissue from the index lesion(s).  This unique tool allows for the most accurate 
alignment of the prostate specimen to MR images that is available today. This resource available 
here at the NCI will be essential for the success of this proposal allowing for precise validation 
of in vivo imaging studies by enabling registration for focal lesion core biopsy and whole mount 
histopathology.
Figure 4: Digitized multiparametric MRI 
maps (left) and the corresponding digitized 
whole-mount histopathology (right) slice with 
tumor outlined by a prostate cancer 
pathologist, and used as a landmark for lesion 
confirmation on mp MRI maps determine 
lesion localization.
1.2.8
Summary
PROSTVAC is a pox viral vaccine in clinical development in prostate cancer.  Preliminary data 
suggest that it can enhance clinical outcomes in advanced prostate cancer and can induce 
systemic immune responses.  A previous study combining the immune checkpoint inhibitor 
ipilimumab and PROSTVAC suggested greater efficacy than PROSTVAC alone.  Additional 
studies have demonstrated the potential of the checkpoint inhibitor nivolumab with the 
possibility of less toxicity.  This study will aim to evaluate the impact of immunologic 
combination therapy on the tumor microenvironment focusing on immune cell infiltration as the 
primary endpoint.  US-MRI imaging technology will be employed to sample the tumor before 
treatment and after radical prostatectomy.  The findings from this study could serve as the basis 
for future studies with combination immunotherapy in this population of patients and more 
advanced disease.

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2
ELIGIBILITY ASSESSMENT AND ENROLLMENT 
2.1
ELIGIBILITY CRITERIA
2.1.1
Inclusion Criteria
For the neoadjuvant cohort, participants must have histopathological documentation of 
adenocarcinoma of the prostate prior to starting this study and evaluable biopsy tissue (e.g., 
unstained slides or blocks) available for analysis. If evaluable tissue is not available, the 
participant must agree to undergo a pre-vaccination prostate biopsy on study.  
For the CRPC lead in cohort, if histopathological documentation is unavailable, a rising PSA 
(see Section 2.1.1.9) and a clinical course consistent with prostate cancer would be acceptable.
2.1.1.1 Age >18 years.  Because no dosing or adverse event data are currently available on the 
use of PROSTVAC  in combination with nivolumab in participants <18 years of age, 
children are excluded from this study, but will be eligible for future pediatric trials.
2.1.1.2 ECOG performance status of 0 or 1 (see Appendix A, Section 16.1).
2.1.1.3 Participants must not have other active invasive malignancies within the past 2 years 
(with the exception of non-melanoma skin cancers) (for CRPC cohort only).
2.1.1.4 Participants must be willing to travel to the study site for follow-up visits
2.1.1.5 All participants who have received prior vaccination with vaccinia virus (for smallpox 
immunization) must not have a history of serious adverse reaction to the vaccine.
2.1.1.6 The effects of PROSTVAC in combination with nivolumab on the developing human 
fetus are unknown.  For this reason men must agree to use adequate contraception 
(abstinence, vasectomy) or female partner must use (intrauterine device (IUD), 
hormonal [birth control, pills, injections, or implants], tubal ligation] prior to study 
entry and for up to 7 months after the last dose. 
2.1.1.7 Participants must understand and sign informed consent that explains the neoplastic 
nature of their disease, the procedures to be followed, the experimental nature of the 
treatment, alternative treatments, potential risks and toxicities, and the voluntary nature 
of participation.
2.1.1.8 Participants must have normal organ and marrow function as defined below: 
a.
hemoglobin
≥ 8 g/dL
b. granulocytes
≥1,500/mcL
c.
platelets
>100,000/mcL
d. total bilirubin
< 1.5 mg/dL (or ≤ 3.0 mg/dL in participants 
with Gilbert syndrome)
e.
AST(SGOT)/ALT(SGPT)
≤ 2.5 X institutional upper limit of normal
f.
creatinine
≤ 1.5 X ULN
2.1.1.9 For the lead in cohort:

Castrate testosterone level (<50ng/dl or 1.7nmol /L)

Progressive disease at study entry defined as one or more of the following criteria 
occurring in the setting of castrate levels of testosterone: 

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
Radiographic progression defined as any new or enlarging bone lesions or growing lymph 
node disease, consistent with prostate cancer 
OR

PSA progression defined by sequence of rising values separated by >1 week (2 separate 
increasing values over a minimum of 2ng/ml (PCWG2 PSA eligibility criteria).  If 
participants had been on flutamide, PSA progression is documented 4 weeks or more after 
withdrawal. For participants on bicalutamide or nilutamide disease progression is 
documented 6 or more weeks after withdrawal.

Participants must agree to continuation of androgen deprivation therapy (ADT) with a 
gonadotropin-releasing hormone agonist/antagonist or bilateral orchiectomy
2.1.1.10 For the neoadjuvant cohort:
1. Participants must be a surgical candidate for radical prostatectomy based on standard 
workup of PSA, biopsy results, and if necessary supplemental imaging. 
2. Participants must have chosen radical prostatectomy as their definitive treatment of 
choice for management of their prostate cancer as part of their standard of care treatment. 
3. No systemic steroid or steroid eye drop use within 2 weeks prior to initiation of 
experimental therapy. Limited doses of systemic steroids to prevent IV contrast, allergic 
reaction or anaphylaxis (in participants who have known contrast allergies) are allowed. 
2.1.2
Exclusion Criteria 
2.1.2.1 Prior splenectomy. 
2.1.2.2 The recombinant vaccinia vaccine should not be administered if the following apply to 
either recipients or, for at least 3 weeks after vaccination, their close household contacts 
(Close household contacts are those who share housing or have close physical contact): 

persons with active or a history of eczema or other eczematoid skin disorders 

those with other acute, chronic or exfoliative skin conditions (e.g., atopic dermatitis, 
burns, impetigo, varicella zoster, severe acne or other open rashes or wounds) until 
condition resolves 

pregnant or nursing women; children under 3 years of age 
2.1.2.3 Participants should have no evidence, as listed below, of being immunocompromised: 

HIV positivity due to the potential for decreased tolerance and risk for severe side effects. 

Hepatitis B or C positivity. 
2.1.2.4 Concurrent use of systemic steroids or steroid eye drops. This is to avoid 
immunosuppression which may lead to potential complications with vaccinia (priming 
vaccination). Nasal, topical or inhaled steroid use is permitted. 
2.1.2.5 Participants with known allergy to eggs or to compounds with a similar chemical or 
biologic composition to PROSTVAC or nivolumab. 
2.1.2.6 No prior immune checkpoint inhibitors (e.g., anti-CTLA4, anti-PD-1 or anti-PDL1 are 
allowed.

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2.1.2.7 Other serious intercurrent illness. 
2.1.2.8 Participants with a history of unstable or newly diagnosed angina pectoris, recent 
myocardial infarction (within 6 months of enrollment) or New York Heart Association 
class II–IV congestive heart failure. 
2.1.2.9 Participants with significant autoimmune disease that is active or potentially life 
threatening if activated. 
2.1.2.10 Participants with clinically significant cardiomyopathy requiring treatment. 
2.1.2.11  Participants with ongoing toxicities related to prior therapies targeting T cell 
coregulatory proteins (immune checkpoints) such as anti-PD-1, anti-PD-L1, or anti-
CTLA-4 antibody are excluded
2.1.2.12 No transfusion of blood or blood products within 2 weeks and no G-CSF or GM-CSF 
within 2 weeks prior to initiations of experimental therapy.
2.1.2.13 Contraindication to biopsy or prostatectomy (for neoadjuvant cohort only):
- Bleeding disorders 
-   Artificial heart valve
- PT/PTT ≥ 1.5 x ULN in participants not taking anticoagulation. Participants on 
anticoagulation (e.g., enoxaparin, oral anticoagulants) are eligible regardless of 
PT/PTT.  Prior to biopsy, anticoagulation will be held per standard practice.
2.1.2.14 For participants with localized prostate cancer contraindication to MRI:
- Participants weighing >136 kilograms (weight limit for the scanner tables)
- Allergy to MR contrast agent
- Participants with pacemakers, cerebral aneurysm clips, shrapnel injury or 
implantable electronic devices
2.1.2.15 History of radiation proctitis (for lead-in CRPC cohort only)
2.1.3
Recruitment Strategies
This protocol may be abstracted into a plain language announcement posted on NIH websites 
and on NIH social media platforms. Participants may also be recruited from the current patient 
population at NIH.
Screening Evaluation
2.1.4
Screening activities performed after a consent for screening has been signed
The following activities will be performed only after the subject has signed the consent for study 
# 01-C-0129 (Eligibility Screening and Tissue Procurement for the NIH Intramural Research 
Program Clinical Protocols) on which screening activities will be performed. Assessments 
performed at outside facilities or on another NIH protocol within the timeframes below may also 
be used to determine eligibility once a participant has signed the consent.
2.1.4.1 For Lead-in CRPC Cohort

Pathological confirmation of diagnosis. May be obtained any time prior to enrollment.

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
The following parameters will be obtained within 8 weeks prior to start of enrollment:
1. HIV test
2. Hepatitis B and C
3. Height

The following parameters will be obtained within 4 weeks prior to start of enrollment:
4. Tc-99 whole-body scintigraphy
5. CT (or MRI may be substituted at investigator’s discretion) of chest, abdomen and 
pelvis

The following parameters will be obtained within 16 days prior to start of enrollment:
6. History and physical examination including vital signs
7. ECOG performance status 
8. Complete blood count plus differential and platelet count 
9. Hepatic (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), Mineral 
(albumin, calcium, magnesium, phosphorus) and Acute Care (sodium, potassium, 
chloride, bicarbonate, creatinine, glucose, BUN, eGFR) Panels
10. Serum lipase
11. Amylase
12. Serum PSA 
13. Testosterone level
14. HIV serology (within 60 days prior to enrollment)
15. Serology for hepatitis B and C (within 60 days prior to enrollment).
16. TSH
17. PT/PTT
2.1.4.2 For neoadjuvant cohort

Pathologic Confirmation of Diagnosis by the Laboratory of Pathology, CC, NIH, or 
Walter Reed National Military Medical Center (if specimen is available). 

Participants that do not have previously collected biopsy material available will be 
asked to undergo a prostate biopsy.  

The following parameters will be obtained within 35 days prior to start of enrollment:

History and Physical Exam, including height measurement 

Performance Status Evaluation 

EKG 

Laboratory Evaluation 

CBC with differential and platelet count 

Acute care panel 

Hepatic Panel 

HIV serology (within 60 days prior to enrollment)

Serology for hepatitis B and C (within 60 days prior to enrollment).

Lipase

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
Amylase

TSH

Serum PSA

PT/PTT
2.2
PARTICIPANT REGISTRATION AND STATUS UPDATE PROCEDURES
Registration and status updates (e.g., when a participant is taken off protocol therapy and when a 
participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant 
Registration & Status Updates found at: 
https://ccrod.cancer.gov/confluence/pages/viewpage.action?pageId=73203825.
2.2.1
Treatment Assignment Procedures
Cohorts
Number
Name
Description
1
Cohort 1
Men with Prostate Cancer
Arms
Number
Name
Description
1
Lead-in CRPC Cohort
PROSTVAC-V on week 0 followed by booster injection called 
PROSTVAC-F on 2, 4, and 8 weeks. Nivolumab will be 
administered every 2 weeks, starting at week 2. When administered 
on the same day, the preferred order of administration is 
PROSTVAC first followed by nivolumab. Participants will undergo 
restaging scans on week 12 prior to receiving treatment.  If no PD, 
option to continue treatment with nivolumab every 2 weeks and 
PROSTVAC-F every 4 weeks until intolerance or progression 
(evaluated radiographically every 12 weeks). Participants who 
remain on protocol beyond 1 year will have the option of extending 
the nivolumab dosing interval to every 4 weeks.  
2
Neoadjuvant Cohort
PROSTVAC-V on week 0 followed by booster injection called 
PROSTVAC-F on 2, 4 and 8 weeks. Nivolumab will be 
administered on weeks 2, 4, 6, and 8. When administered on the 
same day, the preferred order of administration is PROSTVAC first 
followed by nivolumab. Participants will undergo prostatectomy on 
week 9.  (If surgery is scheduled earlier than 9 weeks after initial 
dosing, the 6 and 8 week dosing may skipped and surgery may be 
done as early as week 5.)
Arm Assignment

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Participants in cohort 1 will be assigned to arm 1 until the expected number of participants 
enrolled (n=10), then participants will be assigned to arm 2 (n=17).
2.3
BASELINE EVALUATION
2.3.1
For Lead-In CRPC Cohort
To be performed within 16 days prior to initiation of study therapy (these tests will not need to 
be repeated if they were done at screening within the appropriate timeframe):

History and physical exam including weight and vital signs

ECOG performance status 

CBC with differential and platelet count, prothrombin time/INR, activated partial 
thromboplastin time

HLA class 1 profile (May be obtained any time prior to enrollment)

Urinalysis (may be omitted in participants with incontinence who cannot produce a 
clean sample)

Serum chemistries (Na+, K+, Cl-, CO2, BUN, creatinine, glucose, AST/ALT, 
bilirubin, calcium, phosphorous, albumin, magnesium, alkaline phosphatase, LDH, 
ionized calcium, amylase, lipase, total protein, uric acid)

Serum Testosterone level 

Serum PSA

CPK

Lymphocyte Phenotyping (TBNK) 

Baseline electrocardiogram 12 lead ECG (EKG) on all participants, and appropriate 
cardiologic evaluation, as clinically indicated, to provide baseline function and 
identify any participants who should be monitored closely for cardiac risks associated 
with vaccinia vaccination

Immunology assays (including immune T-cell response assay, CD3, 4, 8 subsets and 
CD4:CD8 ratio, Class II immune responses, CTL assay, and sera antibody analyses) 

Biopsy (May be obtained any time after enrollment and prior to treatment initiation; 
optional)

Scans (CT CAP and bone scan)
2.3.2
For neoadjuvant cohort
To be performed within 16 days prior to enrollment unless specifically stated otherwise (these 
tests will not need to be repeated if they were done at screening within the appropriate 
timeframe):

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
Baseline examinations need not be repeated if they have been performed within the 
appropriate time frame. 

History and Physical Exam 

ECOG Performance Status

HLA class 1 profile (May be obtained any time prior to enrollment)

Serum PSA (tumor marker) 

CBC 

Urinalysis (may be omitted in participants with incontinence who cannot produce a 
clean sample)

Serum chemistries (Na+, K+, Cl-, CO2, BUN, creatinine, glucose, AST/ALT, 
bilirubin, calcium, phosphorous, albumin, magnesium, alkaline phosphatase, LDH, 
ionized calcium, amylase, lipase, total protein, uric acid, C-reactive protein)

Lymphocyte Phenotyping (TBNK) 

Baseline electrocardiogram 12 lead ECG (EKG) on all participants, and appropriate 
cardiologic evaluation, as clinically indicated, to provide baseline function and 
identify any participants who should be monitored closely for cardiac risks associated 
with vaccinia vaccination

Research Studies (See Section 5 for details) 

MRI prostate (can be obtained within one year prior to treatment) MRI of the prostate 
will be performed prior to and after vaccination course if logistically feasible.

Immunology assays (including immune T-cell response assay, CD3, 4, 8 subsets and 
CD4:CD8 ratio, Class II immune responses, CTL assay, and sera antibody analyses) 

Prostate biopsy (performed only if previously collected tissue is not available. If 
biopsy was performed at screening, leftover material may be used for research study.) 
3
STUDY IMPLEMENTATION
3.1
STUDY DESIGN
3.1.1
Rationale for Serial Cohort Design
The primary focus of this study will be to evaluate PROSTVAC and nivolumab in the 
neoadjuvant setting. However, given the concern that treating with these agents prior to radical 
prostatectomy may delay or compromise definitive radical prostatectomy (due to systemic 
adverse events or local inflammation) we will have a lead-in cohort evaluating the safety and 
tolerability of this combination in the castration resistant setting.  
Participants in this initial cohort will be enrolled not more than 3 a week.
Following this lead-in cohort in the CRPC setting, we propose a cohort in the neoadjuvant setting 
evaluating the combination of PROSTVAC and nivolumab. 

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3.1.2
Study Schema
Lead-in CRPC Cohort for safety:
Week 0
Week 2
Week 4
Week 6
Week 8
Week 10
Week 12
N = 
10
PROSTVA
C- V
PROSTVA
C-F
Nivolumab
PROSTVA
C-F
Nivolumab
Nivolum
ab
PROSTVA
C-F
Nivolumab
Nivolumab
Restaging CT 
and Bone scan 
and if no PD 
option to 
continue 
treatment until 
intolerance or 
progression 
(evaluated 
radiographical
ly every 12 
weeks)
PROSTVAC-V 2x108 Infectious Units
PROSTVAC-F 1x109 Infectious Units
Nivolumab 240 mg (Flat dose) every 2 weeks. Alternately, participants who remain on protocol beyond 1 year may 
transition to nivolumab 480 mg (flat dose) every 4 weeks.   
Neoadjuvant Cohort:
Week 0
Week 2
Week 4
Week 6*
Week 8*
Week 9*
N=17
PROSTVAC-V
PROSTVAC
-F
Nivolumab
PROSTVAC
-F
Nivolumab
Nivolumab
PROSTVAC-
F
Nivolumab
Prostatectomy
PROSTVAC-V 2x108 Infectious Units
PROSTVAC-F 1x109 Infectious Units
Nivolumab 240 mg (Flat dose)
*If surgery is scheduled earlier than 9 weeks after initial dosing, the 6 and  8 week dosing may be skipped and 
surgery may be done as early as week 5.
3.2
DRUG ADMINISTRATION
3.2.1
PROSTVAC Vaccine
All vaccines in this trial should be handled according to the guidelines outlined by each 
department.  Study staff administering the vaccine or assessing the vaccine site should wear 
personal protection consistent with the standard of practice outlined by each department or unit.    
Participants receiving PROSTVAC-V should be isolated just prior to the administration of 

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vaccine and can be removed from isolation after the vaccine is administered and bandage is 
secured over the injection site. Appendix B (Section 16.2) defines individuals at risk for vaccinia 
exposure.
PROSTVAC-V (vaccinia) will be administered subcutaneously in an extremity (e.g., thigh) at a 
dose of 2x108 infectious units.
PROSTVAC-F (fowlpox) will be administered subcutaneously in an extremity (e.g., thigh) at a 
dose of 1x109 infectious units.
3.2.2
Nivolumab
Nivolumab is to be administered as a flat dose of 240 mg or 480 mg over approximately 30-
minutes via IV infusion, using a volumetric pump with a 0.2/1.2 micron in-line filter at the 
protocol-specified dose. Nivolumab injection can be infused undiluted or diluted so as not to 
exceed a total infusion volume of 160 mL. Nivolumab may be diluted with 0.9% sodium 
chloride injection, USP or 5% dextrose injection, USP. It is not to be administered as an IV push 
or bolus injection.  At the end of the infusion, flush the line with a sufficient quantity of normal 
saline.
3.2.3
PROSTVAC with Nivolumab 
Toxicity management for the combined agents follows the same template guidelines and 
algorithms that are provided in Section 16.3 for single agent nivolumab.  
When administered on the same day, the preferred order of administration is PROSTVAC first 
followed by nivolumab.
3.3
DOSE MODIFICATIONS
Participants must have recovered to ≤ grade 1 attributable toxicity for the parameters used to 
assess levels of organ function required for eligibility (see Section 2.1) after each treatment in 
order to receive a subsequent treatment. Participants who experience irAE may continue 
PROSTVAC alone if irAE appears to be driven by nivolumab. In such circumstances, 
investigator will discuss fully the potential risks with the participant and document that 
conversation appropriately. Any dosing interruption lasting > 6 weeks from the last dose will 
require participants to be removed from protocol with the following exceptions:

Dosing interruptions to allow for prolonged steroid tapers to manage drug-related
adverse events are allowed. Tumor imaging assessments should continue as per
protocol even if dosing is interrupted

Dosing interruptions > 6 weeks from the last dose that occur for non-drug-related 
reasons may be allowed 

For subjects with mCRPC who have stopped receiving nivolumab because of grade 3 
or higher irAEs, nivolumab could be re-started if the participant had partial response 
according Prostate Cancer Working Group 3 (PCWG 3) and RECIST 1.1 criteria but 
subsequently develops PSA progression (by PCWG 3) while on PROSTVAC alone.
3.4
STOPPING RULES 
The study may be halted if any of the following safety conditions are met:

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
If one or more deaths occur, we will immediately stop accrual and we will promptly discuss 
this with the NIH Intramural IRB and the FDA. In addition, the study will be halted if any 
death occurs within 30 days of treatment regimen.

If two or more participants develop a Grade 4 toxicity at any point in the study attributable to 
the treatment regimen that does not resolve to Grade 2 within 10 days.

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3.5
STUDY CALENDAR
3.5.1
Lead-in CRPC Cohort
Screening 
Week 0 
(Baseline)
Week 2 
(+/- 3 
days)
Week 4
(+/- 3 
days)
Week 6 
(+/- 3 
days)
Week 8 
(+/- 3 
days)
Week 10 
(+/- 3 days)
Follow-up
q 2 weeks until 
off treatment
Safety Visit
4-5 weeks after 
treatment 
discontinuation9
Treatment
Vaccinia-
PROSTVAC
X
Fowlpox-
PROSTVAC
X
X
X
X4
Nivolumab (Flat 
dose)
X
X
X
X
X
X4
Assessments
History and PE, 
Weight
X2
X3
X
X
X
X
X
X4
X
Height
X5
ECOG 
Performance 
Score 
X2
X3
X
X
X
X
X
X4
X
ECG
X3
HIV/Hep B and 
C tests
X5
HLA class I 
profile
X3
Immunology 
Assays/TBNK 
       X
      X3

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Screening 
Week 0 
(Baseline)
Week 2 
(+/- 3 
days)
Week 4
(+/- 3 
days)
Week 6 
(+/- 3 
days)
Week 8 
(+/- 3 
days)
Week 10 
(+/- 3 days)
Follow-up
q 2 weeks until 
off treatment
Safety Visit
4-5 weeks after 
treatment 
discontinuation9
Pathologic 
confirmation of 
dx1
X1
Labs (see 
Sections 2.1.4 
and 2.2.1)
X2
X3
Labs: CBC, 
TSH, amylase, 
lipase, PSA, PT, 
PTT and the 
following 
panels: acute 
care, mineral, 
hepatic 
X
X
X
X
X
X
X
Research labs: 6 
green tops 
(PBMCs) and 2 
SSTs (serum).
X
X
X
X
X7, 8
X
Prostate biopsy 
(optional)
X
Restaging CT 
and Bone scan4
X8
X
Tc 99 
scintigraphy
X6
X

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Screening 
Week 0 
(Baseline)
Week 2 
(+/- 3 
days)
Week 4
(+/- 3 
days)
Week 6 
(+/- 3 
days)
Week 8 
(+/- 3 
days)
Week 10 
(+/- 3 days)
Follow-up
q 2 weeks until 
off treatment
Safety Visit
4-5 weeks after 
treatment 
discontinuation9
CT-C/A/P or 
MRI
X6
X
1Pathologic confirmation will be obtained any time prior to enrollment
2Screening assessments obtained within 16 days prior to enrollment
3Baseline assessments obtained within 16 days prior to initiation of study therapy (these tests will not need to be repeated if they were 
done at screening within the appropriate timeframe)
4If no PD option to continue treatment until intolerance or progression (evaluated radiographically using irRC every 12 weeks). 
PROSTVAC-F is administered every 4 weeks (i.e., weeks 12, 16, 20, etc.) Nivolumab is administered at a flat dose of 240mg every 2 
weeks (+/- 3 days). For participants who remain on protocol beyond 1 year, nivolumab 480mg (flat dose) every 4 weeks (+/- 3 days) 
may be administered instead of the 240 mg every 2-week (+/- 3 days) regimen. 
5To be obtained within 8 weeks prior to start of enrollment
6To be obtained within 4 weeks prior to start of enrollment
7Restaging visits only
8Per PI discretion. For example, participants who have had a complete response to treatment may have the interval between re-staging 
imaging increased to 1 year. Research samples for these participants will also be collected yearly with staging. 
9 Assessment of SAEs at approximately 100 days (± 1 week) after nivolumab discontinuation. Assessment may be conducted by 
phone if the participant cannot come to the NIH. Assessments may also be performed by a local provider.

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3.5.2
Neoadjuvant Cohort
Screening
Week 0
(Baseline)
Week 2 
(+/- 3 
days)
Week 4 
(+/- 3 
days)
Week 66 
(+/- 3 
days)
Week 86 
(+/- 3 days)
Post study 
treatment 
visit 7
Safety Visit
6-8 weeks after treatment 
discontinuation4
Treatment
Vaccinia-
PROSTVAC
X
Fowlpox-
PROSTVAC
X
X
X
Nivolumab 240 
mg (Flat dose)
X
X
X
X
Assessments
History and PE, 
and Weight
X2
X3
X
X
X
X
X
X
Height
X2
ECOG 
Performance 
Score 
X2
X3
X
X
X
X
X
ECG
X2
X3
HLA class I 
profile
X3
Pathologic 
confirmation of 
dx1
X1
Labs (see Sections 
2.1.4 and 2.2.1)
X2
X3
Labs: CBC, TSH, 
X
X
X
X
X
X

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Screening
Week 0
(Baseline)
Week 2 
(+/- 3 
days)
Week 4 
(+/- 3 
days)
Week 66 
(+/- 3 
days)
Week 86 
(+/- 3 days)
Post study 
treatment 
visit 7
Safety Visit
6-8 weeks after treatment 
discontinuation4
amylase, lipase, 
PSA, PT, PTT and 
the following 
panels: acute care, 
mineral, hepatic
Research labs: 6 
green tops 
(PBMCs) and 2 
SSTs (serum).
X
X
X
X
X
Gene expression 
profiling 
(PaxGene tube)
X
X
Immunology 
Assays/TBNK
X
X
X
X
X
X
Prostate biopsy
X
Serum PSA
X
X
X
X
X
X
X
MRI prostate
X
X
PT/PTT/INR 
X
X
1Pathologic confirmation will be obtained any time prior to enrollment
2Screening assessments obtained within 35 days prior to enrollment
3Baseline assessments obtained within 16 days prior to enrollment (these tests will not need to be repeated if they were done at 
screening within the appropriate timeframe)
4Assessment of SAEs at approximately 100 days (± 1 week) after nivolumab discontinuation. Assessment may be conducted by phone 
if the participant cannot come to the NIH. Assessments may also be performed by a local provider.
5A standard of care radical prostatectomy will be performed between week 5 and week 12 week to allow for logistical constraints, but 
ideally as close to week 9 as possible. This may occur at NIH or at an outside institution.

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6If surgery is scheduled earlier than 9 weeks after initial dosing, the 6 and 8 week dosing and study visits/procedures may skipped and 
surgery may be done as early as week 5.
7 This visit should occur one week after the final planned study immunotherapy treatment (-3/+7 days). 

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3.6
COST AND COMPENSATION 
3.6.1
Costs
NIH does not bill health insurance companies or participants for any research or related clinical 
care that participants receive at the NIH Clinical Center.  If some tests and procedures are 
performed outside the NIH Clinical Center, participants may have to pay for these costs .  
Medicines that are not part of the study treatment will not be provided or paid for by the NIH 
Clinical Center.    
3.6.2
Compensation
Participants will not be compensated on this study.
3.6.3
Reimbursement 
The NCI will cover the costs of some expenses associated with protocol participation.  Some of 
these costs may be paid directly by the NIH and some may be reimbursed to the 
participant/guardian as appropriate.  The amount and form of these payments are determined by 
the NCI Travel and Lodging Reimbursement Policy.   
3.7
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA 
Prior to removal from study, effort must be made to have all subjects complete a safety visit 
approximately 4-5 weeks for Lead-in mCRPC cohort and 6-8 weeks for neoadjuvant cohort) 
following the last dose of study therapy and at approximately 100 days after nivolumab 
discontinuation for both cohorts.
3.7.1
Criteria for removal from protocol therapy
Participants will be removed from treatment for the following:

Grade 2 or greater toxicity attributed to treatment that does not resolve to grade 1 within 42 
days from time of scheduled treatment except as described in Section 3.3. 

Grade 2 or greater autoimmune disease that threatens vital organs. 

Intercurrent illness or medical circumstances. If at any time the constraints of this protocol 
are detrimental to the participant’s health, the participant may be removed from protocol 
therapy and reasons for withdrawal will be documented. 

Greater than 3-month delay in the performance of radical prostatectomy from week 12.

Completion of protocol therapy 

Participant requests to be withdrawn from active therapy

Investigator discretion

Clinical or confirmed radiographic progression of disease (for CRPC Cohort only) (irPD)
3.7.2
Off-Study Criteria
1. Patient is off-treatment and has agreed to be followed on a long-term therapy protocol as 
outlined in Section 3.9 or has completed the study follow up period.

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2. The study is stopped.
3. Participant requests to be taken off study. Reasons for withdrawal will be documented.
4. Noncompliance with protocol guidelines (participant removed at discretion of Principal 
Investigator).
5. Disease progression
6. Death 
3.8
FOLLOW-UP EVALUATIONS
After subjects have stopped taking the study medication for any of the reasons listed in Section 
3.6, they will be seen at NIH, if logistically feasible, for a safety visit within 4-5 weeks for Lead-
in mCRPC cohort and 6-8 weeks for neoadjuvant cohort of drug discontinuation.  The safety 
assessments may be performed by a local physician and laboratory if participants unable to 
return to the NIH Clinical Center at this time. 
The following assessments will be performed at the follow up safety visit:

History and Physical Examination

CBC with differential and platelet count, prothrombin time/INR, activated partial 
thromboplastin time

Serum chemistries (Na+, K+, Cl-, CO2, BUN, creatinine, glucose, AST/ALT, bilirubin, 
calcium, phosphorous, albumin, magnesium, alkaline phosphatase, LDH, ionized 
calcium, amylase, lipase, total protein, uric acid), TSH

Serum PSA level

Adverse event reporting  
After the safety visit, if there are no unresolved grade 3 or higher AEs, we will offer participants 
the opportunity to enroll on our long term follow up study.  If there are unresolved grade 3 – 4 
AEs, participants will be followed either at the NIH Clinical Center or by their local physician.  
In the latter case, we will obtain the physician’s record of AEs.
Any scans performed outside of the NIH will also be obtained when possible.  
3.9
POST-STUDY EVALUATION
In 2006, the FDA excluded pox virus based vaccine platforms from required long term follow-
up. Following this guidance in 2006 we haven't built in long term follow-up for our pox-viral 
studies as such vectors have not been shown to have a propensity to integrate or reactivate 
following latency and, in the absence of evidence to the contrary, present a low risk of gene 
therapy-related delayed adverse events. Therefore no long term follow up is required with this 
study, though participants will be offered the opportunity to enroll on our long term follow up 
study.
4
CONCOMITANT MEDICATIONS/MEASURES
Subjects must inform the investigators of the current or planned use or all other medications 
during the study (including prescription medications, vitamins, herbal and nutritional 
supplements, and over-the-counter medications).

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Anti-emetics, stool softeners and antidiarrheal agents may be administered as required but are 
not anticipated to be needed and should not be used prophylactically on the first cycle. The 
selection of the specific antiemetic regimen is at the discretion of the treating physician. 
Antiemetic regimens will not include steroids.
Other supportive care with blood components, antibiotics, analgesics, general medical therapy, 
etc., will be delivered as required. Any participants taking antibiotics for an active infection must 
complete that course of therapy and be free of evidence of further infection before receiving any 
immunotherapy treatment. Antibiotics for prophylaxis are allowed while on treatment. 
Concurrent systemic corticosteroid use (daily or every other day for continued use > 14 days) 
should be avoided within 14 days before the first planned dose of PROSTVAC.  Use of inhaled 
steroids, nasal sprays, and topical creams for small body areas is allowed. Limited use of steroids 
is allowed on study (e.g., for immune related adverse events or for premedication for imaging).
Symptomatic anemia should be treated with appropriate red blood cell or erythropoietin support.
Thrombocytopenia should be treated conservatively. In the absence of bleeding or a planned 
invasive procedure, platelet transfusions should be given for a platelet count below 10,000/mm3. 
If invasive procedures are planned or the participant develops bleeding, platelet transfusions 
should be administered in accordance with the standard of practice, usually maintaining a platelet 
count of > 50,000/mm3.
Any evidence of disseminated intravascular coagulation (DIC), hemolytic uremic syndrome 
(HUS), or thrombotic thrombocytopenic purpura (TTP) including thrombocytopenia, hemolytic 
anemia, renal failure, fever or neurologic changes should be thoroughly evaluated and closely 
monitored and supported as clinically indicated.
A standard of care radical prostatectomy will be performed between week 5 and week 12 to 
allow for logistical constraints, but ideally as close to week 9 as possible. This may occur at NIH 
or at an outside institution. Surgical specimens will be requested for research use (Section 5). 
5
CORRELATIVE STUDIES FOR RESEARCH
5.1
IMMUNE CELL INFILTRATE (TISSUE SPECIMENS)
Slides made from prostate biopsy specimens (collected at baseline) and prostatectomy specimens 
(collected at surgery performed after last dose of vaccine) will be stained for CD4 and CD8 cells 
and digitized for analysis of immune cell infiltrate. Other markers such as CD4/FoxP3 may also 
be stained for further correlative studies. These studies will be performed by the Laboratory of 
Tumor Immunology and Biology. At a minimum, 5 slides from one biopsy core confirmed to 
contain cancer will be needed for baseline quantification. Attempts will be made based on 
anatomic location notation or when available, MRI based location, to correlate biopsy location 
with pathologic location on prostatectomy specimens. When feasible, quantitation will be 
performed in triplicate. Quantitation will be reported as number of stained cells per micron 
squared of surface area. Wilcoxon matched pair signed rank test, Friedman test with Dunn’s 
multiple comparisons, or alternative statistical models will be used as appropriate. Aperio 
ScanScope digital scanner systems with Aperio ImageScope software algorithms will be used. 
Manual quantitation will be performed in case of software malfunction. Isotype antibodies 

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negative controls will be included in all runs. Tonsil or lymph node tissue will be positive control 
for CD4 and CD8. Slides will be developed with primary and secondary antibodies per 
manufacturer specifications for visualization of cells. 
Peripheral Blood Correlative Studies for Research/Pharmacokinetic Studies
Blood Studies 
Blood Tube/Comments** 
Destination 
CBC with differential 
One light lavender tube 
CC Department of Laboratory 
Medicine (DLM) 
Hepatic Panel, Acute Care 
Panel 
One 4 mL SST
 
CC DLM 
Anti-HIV-1/2 
One 8 mL SST 
CC TTV lab 
PT/PTT/INR 
One 4.5 mL citrate blue top 
CC DLM 
HBs Antigen Screening 
Anti-HCV Antibody 
One 8 mL SST 
CC Department of 
Transfusion Medicine (DTM) 
Prostate Specific Antigen 
One 4 mL SST 
CC DLM 
Lymphocyte Phenotyping, 
TBNK 
One light lavender tube 
CC DLM 
Immunology Assays 
Six 10 mL Na Heparin tubes 
Two 8 ml SST tubes 
NCI-Frederick 
1-301-846-5893 
Gene expression profiling*    Two 2.5 mL RNA PaxGene tubes   Blood Processing Core (Figg 
lab)
*RNA PaxGene to be drawn only on the weeks of biopsies or standard of care prostatectomy
** Please note that tubes and media may be substituted based on availability with the permission 
of the PI or laboratory investigator. 
5.1.1
Sera Antibody Analysis 
Serum will be stored at -80 degrees Celsius and there will be planned analysis for generation of 
antibodies to PSA, BCG, PAP, PSMA, PSCA, and/or MUC-1 by the Laboratory of Tumor 
Immunology and Biology. 
5.1.2
PSA-Specific Immune Responses
While many clinical trials have been done in localized prostate cancer, there is little reported on 
the impact of those therapies on the immunologic response that is near the depth and scope that 
is proposed in this trial.  The proposed immunologic studies will be done on all participants by 
the Laboratory of Tumor Immunology and Biology at the NCI Clinical Center under the 
direction of Dr. Jeffrey Schlom.   
Unlike previous studies involving PROSTVAC, which evaluated immune responses to a single 9 
amino acid section of the 244 amino acid PSA protein, the immune analysis in this trial will 
evaluate overlapping 15 amino acid sequences of the entire PSA protein. This more extensive 
analysis will provide a more thorough understanding of the immune response initiated by 

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PROSTVAC and a greater opportunity for clinical correlations.   This type of analysis has not 
been previously prospectively performed in localized prostate cancer participants.  Advantages 
of this approach include the ability to assess both CD4 and CD8 T cells, identification of 
multifunctional T cells (those producing ≥2 cytokines), and identification of T lymphocytes with 
lytic potential (CD107a expression). (Table 1).
  
 
 
 
 
 
A positive “response” will be defined as increased CD107a expression or increased intracellular 
cytokine production for given antigen for CD4 or CD8 cells. For example, Participant #2 in 
Table 1 will be scored has having 5 responses (CD4: INFg, TNF and CD8: CD107a, INFg and 
TNF). 
In order to give added weight to the spread antigens (MUC1 and Brachyury) they will be 
weighted 150% higher than PSA antigens. Thus, the scoring will be as follows: 
For PSA: 
1 point each of the four CD4 parameters (CD107a, IFN-γ, IL-2 and TNF-α) – max total of 4 
1 point each of the four CD8 parameters (CD107a, IFN-γ, IL-2 and TNF-α) – max total of 4 
Total possible score for PSA response is 8 
For MUC1: 
1.5 points each of the four CD4 parameters (CD107a, IFN-γ, IL-2 and TNF-α) – max total of 6 
1.5 points each of the four CD8 parameters (CD107a, IFN-γ, IL-2 and TNF-α) – max total of 6 
Table 1.  Preliminary Data from a trial of Prostvac in mCRPC.  This table shows how this multi 
peptide approach can be used in this proposal.  In this previous (unpublished) trial, 2 cohorts of patients 
were evaluated using the proposed methods.  (Responses are listed as absolute # of CD4 or CD8 T 
Lymphocytes Producing Cytokine or Positive for CD107a per 1x106 cells plated.)  This readout shows 
the breadth of this analysis as both CD4 and CD8 tell cells are evaluated using multiple parameters 
(cytokine production signifying activation – IFN-γ, IL-2 and TNF-α) and lytic potential as measured by 
CD107a expression.  Had only one peptide been used to analyze this immune response it is likely that 
many of these immune responders would not have been identified. (unpublished).

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Total possible score for PSA response is 12. 
For Brachyury: 
1.5 points each of the four CD4 parameters (CD107a, IFN-γ, IL-2 and TNF-α) – max total of 6 
1.5 points each of the four CD8 parameters (CD107a, IFN-γ, IL-2 and TNF-α) – max total of 6 
Total possible score for PSA response is 12.
5.1.3
Flow Cytometry Analysis of Immune subsets (Biomarker Development)
As part of the extensive immune interrogation, peripheral immune cells will be evaluated by 30 
markers to assess 127 immune subsets. (Table 2)  This methodology has been previously 
described [43]. This added analysis will provide preliminary data to develop a Peripheral 
Immunoscore based on the frequency of specific pre-determined immune cell subsets in the 
blood of participants prior to therapy.  The score is calculated from the changes in frequency of 
certain immune cell subsets after vaccine treatment. The resultant panel of markers would reflect 
immune response to the vaccine.  

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Table 2. Analysis of Peripheral Immune Cells
A.
CD4: Helper T lymphocytes (32 subsets)
B.
CD8: Cytotoxic T lymphocytes (29 subsets)
a.
Markers of PD-1 pathway and T cell 
activation (in CD4 and CD8): 
i.
EOMES: activation
ii.
TCR-αβ: activation
iii.
Tbet: activation
iv.
BATF: activation/exhaustion
b.
Maturation status of T lymphocytes (in 
CD4 and CD8): 
i.
Naïve: CD45RA+ CCR7+ 
ii.
Effector Memory: CD45RA- 
CCR7- 
iii.
Terminal (EMRA): CD45RA+ 
CCR7- 
c.
T lymphocyte markers (in CD4 and 
CD8): 
i.
CTLA-4: inhibition 
ii.
PD-1: activation/inhibition 
iii.
PD-L1: activation/cross-
inhibition 
iv.
TIM-3: inhibition 
v.
ICOS: activation (only on 
CD4)
C.
Tregs: Regulatory T lymphocytes (CD4+ CD25+ 
FoxP3+ CD127-) (7 subsets) 
a.
CD45RA: Tregs highly expandable in 
vitro
b.
CTLA-4: Treg suppression
c.
CD49d: “contaminating” effector 
lymphocytes (non-Tregs) 
d.
ICOS: Treg suppression
e.
PD-1: activation/inhibition 
f.
PD-L1: cross-inhibition
D.
B lymphocytes: CD19+ (5 subsets) 
a.
CTLA-4: inhibition 
b.
TIM-3: inhibition 
c.
PD-1: activation/inhibition 
d.
PD-L1: cross-inhibition
E.
NK: Natural killer cells (CD56+ CD3-) (20 subsets) 
a.
CD16+ CD56br: Functional intermediate, 
lytic and cytokine production
b.
CD16+ CD56dim: Mature NK, cytokine 
production
c.
CD16- CD56br: Immature, abundant in 
human placenta
d.
CD16- CD56dim: non-lytic, non-cytokine 
production
e.
TIM-3: activation
f.
PD-1: activation/inhibition 
g.
PD-L1: cross-inhibition
F.
NK-T: CD56+ CD3+ (4 subsets) 
a.
TIM-3: activation
b.
PD-1: activation/inhibition 
c.
PD-L1: cross-inhibition
G.
cDCs (Conventional DCs): CD3-CD56-
CD1c+CD303- (5 subsets) 
H.
pDCs (plasmacytoid DCs ): CD3-CD56-CD1c-
CD303+ (5 subsets) 
a.
Markers of DC activation
i.
CD83: activation 
ii.
TIM-3: inhibition
iii.
PD-1: activation/inhibition 
iv.
PD-L1: cross-inhibition 
I.
MDSCs: Myeloid-derived suppressor cells (CD11b+ 
HLA-DRlow/- CD33+)  (20 subsets) 
a.
CD14: Common Myeloid Marker (high in 
monocytes, dim in granulocytes)
b.
CD15: Granulocyte marker 
c.
CD16: most immature monocytic 
MDSCs
d.
PD-1: activation/inhibition 
e.
PD-L1: cross-inhibition 

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slides and examined, cytokeratin-positive/CD45-negative cells with an intact nucleus and a 
malignancy-consistent morphology are identified as CTCs, and their exact positions on the 
slides recorded. This technology has the advantage of being able to identify cells that may be 
CTC but cytokeratin negative. Since positive or negative selection is not needed, all 
circulating cells are captured and analyzed via proprietary technology, to include multiplex 
analysis.

The evaluation of CTCs may give valuable insight into how treatment affects changes in 
CTC quantity and phenotype, such as AR splice variants, and how such changes are 
associated with clinical outcomes.
Blood collection:
At baseline and at one later timepoint (as close as possible to the time of planned prostatectomy 
or week 9 for initial safety cohort), one 10 mL Streck Cell-Free DNA (brown-black top) tube 
will be collected. Dr. Figg’s lab will be paged at 102-11964 for tube pick up and shipping via 
FedEx Priority Overnight to Epic Sciences. These samples will be sent in ambient shippers 
provided by Epic Sciences to keep samples at room temperature. Alternatively, PBMCs collected 
and stored at -80°C for immunology assays may also be shipped to Epic Sciences for CTC 
analysis. Samples will be shipped to the following address:
Epic Sciences
9381 Judicial Drive, Ste. 200
San Diego, CA  92121
  
5.2
SAMPLE STORAGE, TRACKING AND DISPOSITION
Samples will be ordered in CRIS and tracked through a Clinical Trial Data Management system.  
Should a CRIS screen not be available, the CRIS downtime procedures will be followed.  
Samples will not be sent outside NIH without appropriate approvals and/or agreements, if 
required.
5.2.1
Samples Managed by Dr. Figg’s Blood Processing Core (BPC)
All samples processed by the Blood Processing Core (BPC) will be barcoded, with data entered 
and stored in the Labmatrix utilized by the BPC. This is a secure program, with access to 
Labmatrix limited to defined Figg lab personnel, who are issued individual user accounts. 
Installation of Labmatrix is limited to computers specified by Dr. Figg. These computers all have 
a password restricted login screen. 
Labmatrix creates a unique barcode ID for every sample and sample box, which cannot be traced 
back to participants without Labmatrix access. The data recorded for each sample includes the 
participant ID, name, trial name/protocol number, time drawn, cycle time point, dose, material 
type, as well as box and freezer location. Participant demographics associated with the clinical 
center participant number are provided in the system. For each sample, there are notes associated 
with the processing method (delay in sample processing, storage conditions on the ward, etc.).

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Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80◦C 
according to stability requirements. These freezers are located onsite in the BPC and offsite at 
NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are 
required to be accompanied by laboratory staff at all times. Access to stored clinical samples is 
restricted. Samples will be stored until requested by a researcher named on the protocol. All 
requests are monitored and tracked in Labmatrix. All researchers are required to sign a form 
stating that the samples are only to be used for research purposes associated with this trial (as per 
the IRB approved protocol) and that any unused samples must be returned to the BPC. It is the 
responsibility of the NCI Principal Investigator to ensure that the samples requested are being 
used in a manner consistent with IRB approval.
5.2.2
Samples Sent to Clinical Services Program (CSP)
All data associated with participant samples is protected by using a secure database. All Clinical 
Support Laboratory Staff receive annual training in maintaining records’ confidentiality. All 
samples drawn at the NIH Clinical Center will be transported to the Clinical Support Laboratory 
at the Frederick National Laboratory for Cancer Research by couriers.
Samples will be tracked and managed by the Central Repository database, where there is no link 
to personal identifiable information. All samples will be stored in either a -80˚C freezer or vapor 
phase liquid nitrogen. These freezers are located at NCI Frederick Central Repository in 
Frederick, Maryland.
NCI-Frederick Central Repositories (managed under a subcontract) store, among other things, 
biological specimens in support of NIH clinical studies. All specimens are stored in secure, 
limited access facilities with sufficient security, back up and emergency support capability and 
monitoring to ensure long-term integrity of the specimens for research. 
Specimens are stored in accordance will applicable HHS and FDA Protection of Human Subjects 
Regulations in accordance with the subcontractor’s Federal-wide Assurance.  The 
subcontractor’s role is limited to clinical research databases and repositories containing 
participant specimens. The subcontractor does not conduct nor has any vested interest in research 
on human subjects, but does provide services and support the efforts of its customers, many of 
which are involved in research on human subjects.  The subcontractor’s IRB reviews policies 
and procedures for labeling, data collection and storage, access, and security. The IRB will 
review protection of privacy issues prior to acceptance of any new work and in the event of 
change impacting privacy issues in existing work.
It is the intent and purpose of the subcontractor to accept only coded, linked samples and sample 
information.  To the limit of our ability, every effort will be made to ensure that protected 
information is not sent electronically or by hard copy or on vial labels. 
Sample data is stored in the BioSpecimen Inventory System II (BSI). This inventory tracking 
system is used to manage the storage and retrieval of specimens as well as maintain specimen 
data. BSI is designed for controlled, concurrent access. It provides a real-time, multi-user 
environment for tracking millions of specimens. The system controls how and in what order 
database updates and searches are performed. This control prevents deadlocks and race 
conditions. For security, BSI has user password access, three types of user access levels, and 36 
user permissions (levels of access) that can be set to control access to the system functions. BSI 

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provides audit tracking for processes that are done to specimens including shipping, returning to 
inventory, aliquoting, thawing, additives, and other processes. BSI tracks the ancestry of 
specimens as they are aliquoted, as well as discrepancies and discrepancy resolution for 
specimens received by the repository.  If a specimen goes out of the inventory, the system 
maintains data associated with the withdraw request. Vials are labeled with a unique BSI ID 
which is printed in both eye readable and barcoded format.  No participant specific information 
is encoded in this ID. 
Investigators are granted view, input and withdraw authority only for their specimens. They may 
not view specimen data or access specimens for which they have not been authorized. Access to 
specimen storage is confined to repository staff. Visitors to the repositories are escorted by 
repository staff at all times.
5.2.3
Protocol Completion/Sample Destruction
Once primary research objectives for the protocol are achieved, intramural researchers can 
request access to remaining samples, provided they have an IRB-approved protocol and 
participant consent.
Samples and associated data will be stored permanently unless the participant withdraws 
consent. If the participant withdraws consent the participant’s data will be excluded from future 
distributions, but data that have already been distributed for approved research use will not be 
able to be retrieved. The PI will record any loss or unanticipated destruction of samples as a 
deviation. Reporting will be per the requirements of section 7.2.1.
6
DATA COLLECTION AND EVALUATION 
6.1
DATA COLLECTION
The PI will be responsible for overseeing entry of data into a 21 CFR Part 11- compliant data 
capture system provided by the NCI CCR and ensuring data accuracy, consistency and 
timeliness. The principal investigator, associate investigators/research nurses and/or a contracted 
data manager will assist with the data management efforts. Primary and final analyzed data will 
have identifiers so that research data can be attributed to an individual human subject participant.
All adverse events, including clinically significant abnormal findings on laboratory evaluations, 
regardless of severity, will be followed until return to baseline or stabilization of event.
Document AEs from the first study intervention, Study Day 1, through 30 days after the subject 
received the last study drug administration. Beyond 30 days after the last study drug and through 
approximately 100 days, only adverse events which are serious and related to the study 
intervention need to be recorded.
An abnormal laboratory value will be recorded in the database as an AE only if the laboratory 
abnormality is characterized by any of the following:

Results in discontinuation from the study

Is associated with clinical signs or symptoms 

Requires treatment or any other therapeutic intervention

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
Is associated with death or another serious adverse event, including hospitalization. 

Is judged by the Investigator to be of significant clinical impact
If any abnormal laboratory result is considered clinically significant, the investigator will provide 
details about the action taken with respect to the test drug and about the participant’s outcome.
End of study procedures: Data will be stored according to HHS, FDA regulations and NIH 
Intramural Records Retention Schedule as applicable. 
Loss or destruction of data: Should we become aware that a major breach in our plan to protect 
subject confidentiality and trial data has occurred, this will be reported expeditiously per 
requirements in section 7.2.1. 
-
Eligible participants must be confirmed and checklist completed. Consent form must 
be signed prior to registration with Central Registration Information Services.
-
Data will be secured in study database.  Data will be collected using protocol-specific 
case report forms and verified for accuracy and completeness.  Hard copies of data 
will be stored in locked secured areas and data will be entered onto a secured 
electronic data base. The following protocol-specific study forms will be complete 
and stored: eligibility checklist (developed by Central Registration Office, CRO). A 
copy of all serious AE forms will be kept in the research record.   
-
Treatment is given according to protocol (dated notes about doses given, 
complications, and clinical outcomes).
-
Toxicity is assessed according to protocol (laboratory report slips, etc.)
-
Response is assessed according to protocol (X-ray, scan, lab reports, and date noted 
on clinical assessment, as appropriate).
-
Drug Accountability Records are kept for each participant.
6.2
DATA SHARING PLANS
6.2.1
Human Data Sharing Plan
What data will be shared?
I will share human data generated in this research for future research as follows: 
Coded, linked data in an NIH-funded or approved public repository.  
Coded, linked data in BTRIS (automatic for activities in the Clinical Center)
Identified or coded, linked data with approved outside collaborators under appropriate 
agreements.
How and where will the data be shared? 
Data will be shared through:

An NIH-funded or approved public repository:  clinicaltrials.gov, dbGaP

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
BTRIS (automatic for activities in the Clinical Center)

Approved outside collaborators under appropriate individual agreements.

Publication and/or public presentations.
When will the data be shared? 

Before publication.

At the time of publication or shortly thereafter.
6.2.2
Genomic Data Sharing Plan
Unlinked genomic data will be deposited in public genomic databases such as dbGaP in 
compliance with the NIH Genomic Data Sharing Policy.
6.3
RESPONSE CRITERIA (APPLICABLE TO CRPC COHORT)
Recent clinical studies evaluating checkpoint inhibitors in solid tumors have shown that in a 
fraction of cases even if some new lesions appear at the beginning of a treatment or if the total 
tumor burden does not increase substantially, tumor regressions or stabilizations might still occur 
later. For the CRPC cohort of this trial, a modified immune related response criteria (irRC) based 
on RECIST 1.1 [44] will be used (See Appendix D, Section 16.4). Changes in the largest 
diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the 
case of malignant lymph nodes will be used as per RECIST 1.1 criteria. 
This modified irRC will be based upon RECIST 1.1 criteria (described below) except for 2 major 
changes:
1) require confirmation of progression by imaging at 4 weeks after initial imaging (evidence of 
progression of prostate cancer within the first 3 months on bone scan only should be interpreted 
with extreme caution due to risk of tumor flare) and
2) do not necessarily score the appearance of new lesions as progressive disease if the sum of
lesion diameters of target lesions (minimum of 10 mm per lesion, maximum of 5 target lesions, 
maximum of 2 per organ) and measurable new lesions does not increase by ≥ 20%.
6.3.1
Definitions
Evaluable for toxicity:  All participants will be evaluable for toxicity from the time of their first 
treatment 
Evaluable for objective response: Only those participants who have measurable disease present 
at baseline, have received at least one cycle of therapy, and have had their disease re-evaluated 
will be considered evaluable for response.  These participants will have their response classified 
according to the definitions stated below.  (Note:  Participants who exhibit objective disease 
progression prior to the end of cycle 1 will also be considered evaluable.)
Evaluable Non-Target Disease Response:  Participants who have lesions present at baseline that 
are evaluable but do not meet the definitions of measurable disease, have received at least one 
cycle of therapy, and have had their disease re-evaluated will be considered evaluable for non-

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target disease.  The response assessment is based on the presence, absence, or unequivocal 
progression of the lesions. 
6.3.2
Disease Parameters
Measurable disease:  Measurable lesions are defined as those that can be accurately measured in 
at least one dimension (longest diameter to be recorded) as:

By chest x-ray: >20 mm; 

By CT scan:
.1
Scan slice thickness 5 mm or under: as >10 mm;
.2
Scan slice thickness >5 mm: double the slice thickness

With calipers on clinical exam: >10 mm.  
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters).
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a lymph 
node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness 
recommended to be no greater than 5 mm).  At baseline and in follow-up, only the short axis will 
be measured and followed.
Non-measurable disease.  All other lesions (or sites of disease), including small lesions (longest 
diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), are considered 
non-measurable disease.  Bone lesions, leptomeningeal disease, ascites, pleural/pericardial 
effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses 
(not followed by CT or MRI), are considered as non-measurable.
Note:  Cystic lesions that meet the criteria for radiographically defined simple cysts should not 
be considered as malignant lesions (neither measurable nor non-measurable) since they are, by 
definition, simple cysts.
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable lesions, 
if they meet the definition of measurability described above. However, if non-cystic lesions are 
present in the same participant, these are preferred for selection as target lesions.
Target lesions.  All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in 
total, representative of all involved organs, should be identified as target lesions and recorded 
and measured at baseline.  Target lesions should be selected on the basis of their size (lesions 
with the longest diameter), be representative of all involved organs, but in addition should be 
those that lend themselves to reproducible repeated measurements.  It may be the case that, on 
occasion, the largest lesion does not lend itself to reproducible measurement in which 
circumstance the next largest lesion which can be measured reproducibly should be selected.  A 
sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target 
lesions will be calculated and reported as the baseline sum diameters.  If lymph nodes are to be 
included in the sum, then only the short axis is added into the sum.  The baseline sum diameters 
will be used as reference to further characterize any objective tumor regression in the measurable 
dimension of the disease.

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Non-target lesions.  All other lesions (or sites of disease) including any measurable lesions over 
and above the 5 target lesions should be identified as non-target lesions and should also be 
recorded at baseline.  Measurements of these lesions are not required, but the presence, absence, 
or in rare cases unequivocal progression of each should be noted throughout follow-up. 
6.3.3
Methods for Evaluation of Measurable Disease
All measurements should be taken and recorded in metric notation using a ruler or calipers.  All 
baseline evaluations should be performed as closely as possible to the beginning of treatment and 
never more than 8 weeks before the beginning of the treatment.
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam.
Clinical lesions:  Clinical lesions will only be considered measurable when they are superficial 
(e.g., skin nodules and palpable lymph nodes) and 10 mm diameter as assessed using calipers 
(e.g., skin nodules).  In the case of skin lesions, documentation by color photography, including a 
ruler to estimate the size of the lesion, is recommended. 
Chest x-ray:  Lesions on chest x-ray are acceptable as measurable lesions when they are clearly 
defined and surrounded by aerated lung.  However, CT is preferable. 
Conventional CT and MRI:  This guideline has defined measurability of lesions on CT scan 
based on the assumption that CT slice thickness is 5 mm or less.  If CT scans have slice thickness 
greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness.  
MRI is also acceptable in certain situations (e.g. for body scans).  
Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which greatly 
impact image quality, lesion conspicuity, and measurement.  Furthermore, the availability of 
MRI is variable globally.  As with CT, if an MRI is performed, the technical specifications of the 
scanning sequences used should be optimized for the evaluation of the type and site of disease.  
Furthermore, as with CT, the modality used at follow-up should be the same as was used at 
baseline and the lesions should be measured/assessed on the same pulse sequence.  It is beyond 
the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all 
scanners, body parts, and diseases.  Ideally, the same type of scanner should be used and the 
image acquisition protocol should be followed as closely as possible to prior scans.  Body scans 
should be performed with breath-hold scanning techniques, if possible.
PET-CT:  At present, the low dose or attenuation correction CT portion of a combined PET-CT 
is not always of optimal diagnostic CT quality for use with RECIST measurements.  However, if 
the site can document that the CT performed as part of a PET-CT is of identical diagnostic 
quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be 
used for RECIST measurements and can be used interchangeably with conventional CT in 
accurately measuring cancer lesions over time.  Note, however, that the PET portion of the CT 
introduces additional data which may bias an investigator if it is not routinely or serially 
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Ultrasound:  Ultrasound is not useful in assessment of lesion size and should not be used as a 
method of measurement.  Ultrasound examinations cannot be reproduced in their entirety for 
independent review at a later date and, because they are operator dependent, it cannot be 
guaranteed that the same technique and measurements will be taken from one assessment to the 
next.  If new lesions are identified by ultrasound in the course of the study, confirmation by CT 
or MRI is advised.  If there is concern about radiation exposure at CT, MRI may be used instead 
of CT in selected instances.
Endoscopy, Laparoscopy:  The utilization of these techniques for objective tumor evaluation is 
not advised.  However, such techniques may be useful to confirm complete pathological 
response when biopsies are obtained or to determine relapse in trials where recurrence following 
complete response (CR) or surgical resection is an endpoint.
Tumor markers:  Tumor markers alone cannot be used to assess response.  If markers are initially 
above the upper normal limit, they must normalize for a participant to be considered in complete 
clinical response.  Specific guidelines for both CA-125 response (in recurrent ovarian cancer) 
and PSA response (in recurrent prostate cancer) have been published. [45-47] In addition, the 
Gynecologic Cancer Intergroup has developed CA-125 progression criteria which are to be 
integrated with objective tumor assessment for use in first-line trials in ovarian cancer.[48]
Cytology, Histology:  These techniques can be used to differentiate between partial responses 
(PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as 
germ cell tumors, where known residual benign tumors can remain).
The cytological confirmation of the neoplastic origin of any effusion that appears or worsens 
during treatment when the measurable tumor has met criteria for response or stable disease is 
mandatory to differentiate between response or stable disease (an effusion may be a side effect of 
the treatment) and progressive disease.
FDG-PET:  While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease).  New lesions on the basis of 
FDG-PET imaging can be identified according to the following algorithm: 

Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD 
based on a new lesion.

No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive FDG-
PET at follow-up corresponds to a new site of disease confirmed by CT, this is PD.  If the 
positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, 
additional follow-up CT  scans are needed to determine if there is truly progression 
occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-
PET scan).  If the positive FDG-PET at follow-up corresponds to a pre-existing site of 
disease on CT that is not progressing on the basis of the anatomic images, this is not PD.

FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in 
cases where a residual radiographic abnormality is thought to represent fibrosis or 
scarring.  The use of FDG-PET in this circumstance should be prospectively described in 
the protocol and supported by disease-specific medical literature for the indication.  
However, it must be acknowledged that both approaches may lead to false positive CR 
due to limitations of FDG-PET and biopsy resolution/sensitivity.

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 Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake greater 
than twice that of the surrounding tissue on the attenuation corrected image.
6.3.4
Response Criteria
6.3.4.1 Evaluation of Target Lesions
Complete Response (CR): Disappearance of all target lesions.  Any pathological lymph nodes 
(whether target or non-target) must have reduction in short axis to <10 mm.
Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, 
taking as reference the baseline sum of diameters.
Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, 
taking as reference the smallest sum on study (this includes the baseline sum if that is the 
smallest on study).  In addition to the relative increase of 20%, the sum must also demonstrate an 
absolute increase of at least 5 mm.  (Note:  the appearance of one or more new lesions is also 
considered progressions).
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to 
qualify for PD, taking as reference the smallest sum of diameters while on study.
6.3.4.2 Evaluation of Non-Target Lesions
Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor 
marker level.  All lymph nodes must be non-pathological in size (<10 mm short axis).
Note:  If tumor markers are initially above the upper normal limit, they must normalize for a 
participant to be considered in complete clinical response.
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor 
marker level above the normal limits.
Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal 
progression of existing non-target lesions.  Unequivocal progression should not normally trump 
target lesion status.  It must be representative of overall disease status change, not a single lesion 
increase.    
Although a clear progression of “non-target” lesions only is exceptional, the opinion of the 
treating physician should prevail in such circumstances, and the progression status should be 
confirmed at a later time by the review panel (or Principal Investigator).
6.3.4.3 Metastatic Bone Lesions
Disease progression is considered if a minimum of two new lesions is observed on bone scan.  
6.3.4.4 Evaluation of Best Overall Response
The best overall response is the best response recorded from the start of the treatment until 
disease progression/recurrence (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started).  The participant's best response assignment 
will depend on the achievement of both measurement and confirmation criteria.
For Participants with Measurable Disease (i.e., Target Disease)

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Target 
Lesions
Non-Target 
Lesions
New 
Lesions
Overall 
Response
Best Overall Response when 
Confirmation is Required*
CR
CR
No
CR
>4 wks. Confirmation**
CR
Non-
CR/Non-PD
No
PR
CR
Not 
evaluated
No
PR
PR
Non-
CR/Non-
PD/not 
evaluated
No
PR
>4 wks. Confirmation**
SD
Non-
CR/Non-
PD/not 
evaluated
No
SD
Documented at least once >4 
wks. from baseline**
PD
Any
Yes or 
No
PD
Any
PD***
Yes or 
No
PD
Any
Any
Yes
PD
no prior SD, PR or CR
*
See RECIST 1.1 manuscript for further details on what is evidence of a 
new lesion.
**
Only for non-randomized trials with response as primary endpoint.
***
In exceptional circumstances, unequivocal progression in non-target 
lesions may be accepted as disease progression.
Note: Participants with a global deterioration of health status requiring 
discontinuation of treatment without objective evidence of disease 
progression at that time should be reported as “symptomatic 
deterioration.”  Every effort should be made to document the objective 
progression even after discontinuation of treatment.
For Participants with Non-Measurable Disease (i.e., Non-Target Disease)
Non-Target Lesions
New Lesions
Overall Response

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6.3.5
Duration of Response
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first date that 
recurrent or progressive disease is objectively documented (taking as reference for progressive 
disease the smallest measurements recorded since the treatment started).
The duration of overall CR is measured from the time measurement criteria are first met for CR 
until the first date that progressive disease is objectively documented.
Duration of stable disease:  Stable disease is measured from the start of the treatment until the 
criteria for progression are met, taking as reference the smallest measurements recorded since the 
treatment started, including the baseline measurements. 
6.3.6
Progression-Free Survival
6.3.6.1 Serologic Response
PSA will be assessed at each visit during treatment and every 12 weeks thereafter.  Participants 
will be assigned response based on the following criteria.
Complete Serological Response:  PSA level less than 0.2 ng/ml measured for 2 consecutive 
measurements at least 4 weeks apart.
Serological Partial Response:  decline of PSA at least 50% measured for 2 consecutive 
measurements at least 4 weeks apart.
Serological Progression:  

Serological progression will only be measured once PSA has risen above 4 ng/ml 
and this value must be 50% above the PSA level before commencing ADT and;

Increase in PSA more than 50% of nadir (lowest PSA on ADT).  

Values must be measured for 2 consecutive measurements at least 2 weeks apart.  

The date of the first increase will be recorded as progression.  
CR
No
CR
Non-CR/non-PD
No
Non-CR/non-PD*
Not all evaluated
No
not evaluated
Unequivocal PD
Yes or No
PD
Any
Yes
PD
*  ‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease 
since SD is increasingly used as an endpoint for assessment of efficacy in some 
trials so to assign this category when no lesions can be measured is not advised

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Time to Serologic Progression will be defined as interval between initiation of ADT and date of 
PSA progression or death from any cause.
6.4
TOXICITY CRITERIA
The following adverse event management guidelines are intended to ensure the safety of each 
participant while on the study.  The descriptions and grading scales found in the revised NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE 
reporting.  All appropriate treatment areas should have access to a copy of the CTCAE version 
4.0.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm).  
7
NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
7.1
DEFINITIONS
Please refer to definitions provided in Policy 801: Reporting Research Events found at: 
https://irbo.nih.gov/confluence/pages/viewpage.action?pageId=36241835#Policies&Guidance-
800Series-ComplianceandResearchEventReportingRequirements.
7.2
OHSRP OFFICE OF COMPLIANCE AND TRAINING/  IRB  REPORTING
7.2.1
 Expedited Reporting
Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 
802 Non-Compliance Human Subjects Research found at: 
https://irbo.nih.gov/confluence/pages/viewpage.action?pageId=36241835#Policies&Guidance-
800Series-ComplianceandResearchEventReportingRequirements. Note: Only IND Safety 
Reports that meet the definition of an unanticipated problem will need to be reported per these 
policies.
7.2.2
IRB Requirements for PI Reporting at Continuing Review
Please refer to the reporting requirements in Policy 801: Reporting Research Events found at: 
https://irbo.nih.gov/confluence/pages/viewpage.action?pageId=36241835#Policies&Guidance-
800Series-ComplianceandResearchEventReportingRequirements. 
7.3
NCI CLINICAL DIRECTOR REPORTING
Problems expeditiously reviewed by the OHSRP in the NIH eIRB system  will also be reported 
to the NCI Clinical Director/designee; therefore, a separate submission for these reports is not 
necessary.  
In addition to those reports, all deaths that occur within 30 days after receiving a research 
intervention should be reported via email unless they are due to progressive disease.  
To report these deaths, please send an email describing the circumstances of the death to 
NCICCRQA@mail.nih.gov within one business day of learning of the death. 

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7.4
INSTITUTIONAL BIOSAFETY COMMITTEE (IBC) REPORTING CRITERIA
7.4.1
Serious Adverse Event Reports to IBC
The Principal Investigator (or delegate) will notify IBC of any unexpected fatal or life-
threatening experience associated with the use of PROSTVAC as soon as possible but in no 
event later than 7 calendar days of initial receipt of the information.  Serious adverse events that 
are unexpected and associated with the use of the PROSTVAC, but are not fatal or life-
threatening, must be reported to the NIH IBC as soon as possible, but not later than 15 calendar 
days after the investigator’s initial receipt of the information.  Adverse events may be reported 
by using the FDA Form 3500a.
7.4.2
Annual Reports to IBC
Within 60 days after the one-year anniversary of the date on which the IBC approved the initial 
protocol, and after each subsequent anniversary until the trial is completed, the Principal 
Investigator (or delegate) shall submit the information described below.  Alternatively, the IRB 
continuing review report can be sent to the IBC in lieu of a separate report.  Please include the 
IBC protocol number on the report.
7.4.3
Clinical Trial Information 
A brief summary of the status of the trial in progress or completed during the previous year. The 
summary is required to include the following information: 

the title and purpose of the trial 

clinical site

the Principal Investigator

clinical protocol identifiers; 

participant population (such as disease indication and general age group, e.g., adult or 
pediatric); 

the total number of participants planned for inclusion in the trial; the number entered into 
the trial to date whose participation in the trial was completed; and the number who 
dropped out of the trial with a brief description of the reasons 

the status of the trial, e.g., open to accrual of subjects, closed but data collection ongoing, 
or fully completed, 

if the trial has been completed, a brief description of any study results. 
7.4.4
Progress Report and Data Analysis 
Information obtained during the previous year's clinical and non-clinical investigations, 
including: 

a narrative or tabular summary showing the most frequent and most 
serious adverse experiences by body system

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
a summary of all serious adverse events submitted during the past year

a summary of serious adverse events that were expected or considered to 
have causes not associated with the use of the gene transfer product such 
as disease progression or concurrent medications

if any deaths have occurred, the number of participants who died during 
participation in the investigation and causes of death

a brief description of any information obtained that is pertinent to an 
understanding of the gene transfer product’s actions, including, for 
example, information about dose-response, information from controlled 
trials, and information about bioavailability. 
7.5
NIH REQUIRED DATA AND SAFETY MONITORING PLAN
7.5.1
Principal Investigator/Research Team 
The clinical research team will meet on a regular basis (approximately weekly) when participants 
are being actively treated on the trial to discuss each participant. Decisions about dose level 
enrollment and dose escalation if applicable will be made based on the toxicity data from prior 
participants. 
All data will be collected in a timely manner and reviewed by the principal investigator or a lead 
associate investigator. 
Events meeting requirements for expedited reporting as described in section Error! Reference 
source not found. will be submitted within the appropriate timelines.  
The principal investigator will review adverse event and response data on each participant to 
ensure safety and data accuracy. The principal investigator will personally conduct or supervise 
the investigation and provide appropriate delegation of responsibilities to other members of the 
research staff. 
8
SPONSOR PROTOCOL/SAFETY REPORTING
8.1
DEFINITIONS
8.1.1
Adverse Event 
Any untoward medical occurrence in a participant or clinical investigation subject administered a 
pharmaceutical product and which does not necessarily have a causal relationship with this 
treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally associated with the 
use of a medicinal (investigational) product, whether or not related to the medicinal 
(investigational) product (ICH E6 (R2))

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8.1.2
Serious Adverse Event (SAE)
An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in any of the following:

Death,

A life-threatening adverse event (see section 8.1.3)

Inpatient hospitalization or prolongation of existing hospitalization 
o
A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery 
arranged prior to the start of the study), a planned hospitalization for pre-existing 
condition, or a procedure required by the protocol, without a serious deterioration in 
health, is not considered a serious adverse event.
o
A hospitalization/admission that is solely driven by non-medical reasons (e.g., 
hospitalization for patient convenience) is not considered a serious adverse event. 
o
Emergency room visits or stays in observation units that do not result in admission to 
the hospital would not be considered a serious adverse event. The reason for seeking 
medical care should be evaluated for meeting one of the other serious criteria.

Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions

A congenital anomaly/birth defect

Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition.
8.1.3
Life-threatening 
An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of 
either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of 
death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a 
more severe form, might have caused death. (21CFR312.32)
8.1.4
Severity
The severity of each Adverse Event will be assessed utilizing the CTCAE version 4.0.
8.1.5
Relationship to Study Product
All AEs will have their relationship to study product assessed using the terms:  related or not 
related.  

Related – There is a reasonable possibility that the study product caused the adverse 
event. Reasonable possibility means that there is evidence to suggest a causal relationship 
between the study product and the adverse event.

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
Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event.
8.2
ASSESSMENT OF SAFETY EVENTS
AE information collected will include event description, date of onset, assessment of severity 
and relationship to study product and alternate etiology (if not related to study product), date of 
resolution of the event, seriousness and outcome. The assessment of severity and relationship to 
the study product will be done only by those with the training and authority to make a diagnosis 
and listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  AEs 
occurring during the collection and reporting period will be documented appropriately regardless 
of relationship.  AEs will be followed through resolution.
SAEs will be:

Assessed for severity and relationship to study product and alternate etiology (if not 
related to study product) by a licensed study physician listed on the Form FDA 1572 as 
the site principal investigator or sub-investigator.

Recorded on the appropriate SAE report form, the medical record and captured in the 
clinical database.

Followed through resolution by a licensed study physician listed on the Form FDA 1572 
as the site principal investigator or sub-investigator.
For timeframe of recording adverse events, please refer to section 6.1. All serious adverse events 
recorded from the time of first investigational product administration must be reported to the 
sponsor. 
8.3
REPORTING OF SERIOUS ADVERSE EVENTS
Any AE that meets protocol-defined serious criteria or meets the definition of Adverse Event of 
Special Interest that require expedited reporting must be submitted immediately (within 24 hours 
of awareness) to OSRO Safety using the CCR SAE report form.  All SAE reporting must include 
the elements described in section 8.2.
SAE reports will be submitted to the Center for Cancer Research (CCR) at: 
OSROSafety@mail.nih.gov and to the CCR PI and study coordinator.  CCR SAE report form 
and instructions can be found at: 
https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions
Following the assessment of the SAE by OSRO, other supporting documentation of the event 
may be requested by the OSRO Safety and should be provided as soon as possible.
8.4
SAFETY REPORTING CRITERIA TO THE PHARMACEUTICAL COLLABORATORS
All events listed below must be reported in the defined timelines to OSROSafety@mail.nih.gov. 
The CCR Office of Sponsor and Regulatory Oversight (OSRO) will send all reports to the 
manufacturer as described below.

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8.4.1
Bavarian-Nordic
To be sent by Office of Sponsor and Regulatory Oversight (OSRO), CCR, NCI
The Investigator should complete and submit an SAE Medwatch 3500 Form or an CCR SAE 
report form to OSRO containing all information that is required by the Regulatory Authorities, to 
BNIT by e-mail within 24 to 48 hours of awareness for SAEs deemed to be possibly, probably or 
definitely related to the study vaccines. 
The SAE documentation, including the Medwatch 3500 Form or CCR SAE report form and 
available source records, should be emailed to: 
Bavarian-Nordic, Inc.
Email: drug.safety@bavarian-nordic.com
The following minimum information is required: 
o
Study number/IIT regulatory identifier 
o
Subject number, sex and age 
o
The date of report 
o
A description of the SAE (event, seriousness of the event) 
o
Causal relationship to the study drug 
Follow-up information for the event should be sent within 7 days as necessary. 
SAEs that are deemed unrelated or unlikely to be due to the study vaccines are submitted 
regularly to BNIT at the following address: 
Bavarian-Nordic, Inc.
Email: drug.safety@bavarian-nordic.com
8.4.2
Bristol-Myers-Squibb
To be sent by OSRO, CCR, NCI, NIH: 
Following the subject’s written consent to participate in the study, all SAEs, whether related or 
not related to study drug, must be collected, including those thought to be associated with 
protocol-specified procedures. All SAEs must be collected that occur within 100 days of 
discontinuation of dosing. 
All SAEs must be collected that occur during the screening period. If applicable, SAEs must be 
collected that relate to any protocol-specified procedure (e.g., a follow-up skin biopsy).  The 
investigator should report any SAE that occurs after these time periods that is believed to be 
related to study drug or protocol-specified procedure. 
SAEs, whether related or not related to study drug, and pregnancies must be reported to BMS 
within 24 hours. SAEs must be recorded on BMS or an approved form; pregnancies on a 
Pregnancy Surveillance Form.
SAE Email Address:  Worldwide.Safety@BMS.com

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SAE Facsimile Number:  609-818-3804
If only limited information is initially available, follow-up reports are required. (Note: Follow-up 
SAE reports should include the same investigator term(s) initially reported.) 
If an ongoing SAE changes in its intensity or relationship to study drug or if new information 
becomes available, a follow-up SAE report should be sent within 24 hours to the BMS (or 
designee) using the same procedure used for transmitting the initial SAE report. 
All SAEs should be followed to resolution or stabilization.
The Investigator will ensure that all SAEs in the clinical database are reported to BMS and any 
applicable health authority during the conduct of the study. This reconciliation will occur at least 
quarterly and be initiated by the investigator. Investigator will request a reconciliation report 
from: aepbusinessprocess@bms.com. During reconciliation, any events found to not be reported 
previously to BMS must be sent to Worldwide.Safety@BMS.com. 
BMS will be provided with a simultaneous copy of all adverse events filed with the FDA. 
SAEs should be reported on MedWatch Form 3500A, which can be accessed at: 
http://www.accessdata.fda.gov/scripts/medwatch/ or CCR SAE report form.
All SAEs should simultaneously be faxed or e-mailed to BMS at:
Global Pharmacovigilance & Epidemiology
Bristol-Myers Squibb Company
Fax Number: 609-818-3804
Email: Worldwide.safety@bms.com
8.5
REPORTING PREGNANCY 
All required pregnancy reports/follow-up to OSRO will be submitted to: 
OSROSafety@mail.nih.gov and to the CCR PI and study coordinator.  Forms and instructions 
can be found here: 
https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions 
8.5.1
Paternal exposure
Male participants should refrain from fathering a child or donating sperm during the study and 
for 7 months after the last dose of study agent.
Pregnancy of the participant’s partner is not considered to be an AE. However, the outcome of 
all pregnancies occurring from the date of the first dose until 7 months after the last dose should, 
if possible, be followed up and documented. Pregnant partners may be offered the opportunity to 
participate in an institutional pregnancy registry protocol (e.g., the NIH IRP pregnancy registry 
study) to provide data about the outcome of the pregnancy for safety reporting purposes.
8.6
REGULATORY REPORTING FOR STUDIES CONDUCTED UNDER CCR-SPONSORED IND
Following notification from the investigator, CCR, the IND sponsor, will report any suspected 
adverse reaction that is both serious and unexpected.  CCR will report an AE as a suspected 
adverse reaction only if there is evidence to suggest a causal relationship between the study 

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product and the adverse event.  CCR will notify FDA and all participating investigators (i.e., all 
investigators to whom the sponsor is providing drug under its INDs or under any investigator’s 
IND) in an IND safety report of potential serious risks from clinical trials or any other source, as 
soon as possible, in accordance to 21 CFR Part 312.32. 
All serious events will be reported to the FDA at least annually in a summary format.
8.7
SPONSOR PROTOCOL DEVIATION REPORTING
A Protocol Deviation is defined as any non-compliance with the clinical trial Protocol, Manual 
of Operational Procedures (MOP) and other Sponsor approved study related documents, GCP, or 
protocol-specific procedural requirements on the part of the participant, the Investigator, or the 
study site staff inclusive of site personnel performing procedures or providing services in support 
of the clinical trial.
It is the responsibility of the study Staff to document any protocol deviation identified by the 
Staff or the site Monitor in the CCR Protocol Deviation Tracking System (PDTS) online 
application. The entries into the PDTS online application should be timely, complete, and 
maintained per CCR PDTS user requirements. 
In addition, any deviation to the protocol should be documented in the participant’s source 
records and reported to the reviewing IRB per their guidelines. OSRO required protocol 
deviation reporting is consistent with E6(R2) GCP: Integrated Addendum to ICH E6(R1): 4.5 
Compliance with Protocol; 5.18.3 (a), and 5.20 Noncompliance; and ICH E3 16.2.2 Protocol 
deviations.
9
CLINICAL MONITORING 
Clinical site monitoring is conducted to ensure:

that the rights of the participants are protected; 

that the study is implemented per the approved protocol, Good Clinical Practice and 
standard operating procedures; and, 

the quality and integrity of study data and data collection methods are maintained.  
Monitoring for this study will be performed by NCI CCR Office of Sponsor and Regulatory 
Oversight (OSRO) Sponsor and Regulatory Oversight Support (SROS) Services contractor. 
Clinical site monitoring activities will be based on OSRO standards, FDA Guidance E6(R2) 
Good Clinical Practice: Integrated Addendum to ICH E6(R1) March 2018, and applicable 
regulatory requirements.
Details of clinical site monitoring will be documented in a Clinical Monitoring Plan (CMP) 
developed by OSRO. CMPs will be protocol-specific, risk-based and tailored to address human 
subject protections and integrity of the study data. OSRO will determine the intensity and 
frequency of monitoring based on several factors, including study type, phase, risk, complexity, 
expected enrollment rate, and any unique attributes of the study and the site. The Sponsor will 
conduct a periodic review of the CMP to confirm the plan's continued appropriateness. A change 
to the protocol, significant or pervasive non-compliance with GCP, or the protocol may trigger 
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OSRO SROS Monitoring visits and related activities will be conducted throughout the life cycle 
of each protocol. The first activity is before the study starts to conduct a Site Assessment Visit 
(SAV) (as warranted), followed by a Site Initiation Visit (SIV), Interim Monitoring Visit(s) 
(IMVs), and a study Close-Out Visit (COV).
Some monitoring activities may be performed remotely, while others will occur at the study 
site(s). Monitoring visit reports will describe visit activities, observations, and associated action 
items or follow-up required for resolution of any issues, discrepancies, or deviations. Monitoring 
reports will be distributed to the study PI, NCI CCR QA, CCR Protocol Support Office, 
coordinating center (if applicable), and the Sponsor regulatory file.
The site Monitor will inform the study team of any deviations observed during monitoring visits. 
If unresolved, the Monitor will request that the site Staff enter the deviations in the CCR 
Protocol Deviation Tracking System (PDTS) for deviation reporting to the Sponsor and as 
applicable per institutional and IRB guidance.
10 STATISTICAL CONSIDERATIONS  
For the initial lead-in cohort, the primary objective is safety, especially detection of clinically 
important inflammation defined as a grade 3 diarrhea or colitis requiring steroids or anti cytokine 
therapy or not resolving to grade 1 or less within 28 days. If there are 10 participants evaluated in 
the lead-in cohort and if 0/10 have clinically important inflammation, then this will be 
considered acceptable for proceeding with the neoadjuvant cohort. On the other hand, if 1/10 or 
more have significant inflammation, then we will discuss with the IRB, FDA and sponsor 
amending the trial, since the upper one-sided 90% confidence interval on 1/10 is 33.7% which 
would be consistent with an excessive rate of inflammation.
After the lead-in cohort, participants on this study will be assigned to PROSTVAC and 
Nivolumab (neoadjuvant cohort) for 9 weeks. Utilizing computer automated staining analysis, 
density of cell infiltrate will be calculated and the pre- and post-treatment values will be 
compared to determine response to treatment. All measurement will be determined in a blinded 
fashion with respect to the treatment groups.  The primary evaluation will be of a comparison of 
differences between the neoadjuvant cohort and participants treated on the 14-C-0112 protocol 
(NCT02153918), of the changes of the CD3 + T cells, measured from baseline to the radical 
prostatectomy at 9 weeks on the present trial, and from baseline to the radical prostatectomy at 
12 weeks in participants treated on the 14-C-0112 trial.
With 17 total evaluable participants having both  CD3+/CD4+ and CD3+/CD8+ T cell 
measurements from baseline to 9 weeks on the present trial, and comparable data from 26 
participants on the 14-C-0112 trial, there would be adequate participants to have 80% power to 
detect a difference between the cohort from this trial and that from the prior trial of a change in 
each type of CD3+ T cells from baseline to 9 weeks  (or baseline to 12 weeks in the 14-C-0112 
trial) equal to 1.0 SD of the change in the CD3 + T cells (1.0 SD effect size) using a 0.025 
significance level two-tailed two group t-test to allow adequate participants for a Bonferroni 
adjustment holding the overall significance level to 0.05 for both tests. In practice, a Hochberg 
adjustment may be used instead of the overly stringent Bonferroni adjustment. Also, if the 
distributions of the CD3 + T cells are not normally distributed (p<0.05 by a Shapiro-Wilks test), 
then a Wilcoxon rank sum test may be used instead of a two-sample t-test. The results from the 

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comparison of data from the two studies will be reported with appropriate caveats to reflect 
differences in study characteristics, and to reflect the lack of randomized participant assignment 
between the two trials.
Secondary objectives are to determine the change in peripheral PSA-specific T cells in 
participants treated with these vaccines; to document any intraprostatic Treg cell infiltration with 
CD4+FOX-P3 staining; to document any PSA changes secondary to vaccination, including rate 
of biochemical recurrence after prostatectomy; to document any MRI changes secondary to 
treatment; to evaluate changes in PDL-1 expression; and to document pathologic responses 
(including pathologic CR). When analysis is required, each of these endpoints will be analyzed 
using appropriate nonparametric methods when applicable and the results from these analyses 
will be presented without formal adjustment for multiple comparisons, but in the context of the 
number of such evaluations undertaken.
Slides made from prostate biopsy specimens (collected at baseline) and prostatectomy specimens 
(collected at surgery performed after last dose of vaccine) will be stained for CD4 and CD8 cells 
and digitized for analysis of immune cell infiltrate. Other markers such as CD4/FoxP3 may also 
be stained for further correlative studies. These studies will be performed by the Laboratory of 
Tumor Immunology and Biology. At a minimum, 5 slides from one biopsy core confirmed to 
contain cancer will be needed for baseline quantification. Attempts will be made based on 
anatomic location notation or when available, MRI based location, to correlate biopsy location 
with pathologic location on prostatectomy specimens. When feasible, quantitation will be 
performed in triplicate. Quantitation will be reported as number of stained cells per micron 
squared of surface area. Wilcoxon matched pair signed rank test, Friedman test with Dunn’s 
multiple comparisons, or alternative statistical models will be used as appropriate. Aperio 
ScanScope digital scanner systems with Aperio ImageScope software algorithms will be used. 
Manual quantitation will be performed in case of software malfunction. Isotype antibodies 
negative controls will be included in all runs. Tonsil or lymph node tissue will be positive control 
for CD4 and CD8. Slides will be developed with primary and secondary antibodies per 
manufacturer specifications for visualization of cells. 
The lead-in safety cohort will require 10 participants and the neoadjuvant cohort will require 17 
evaluable participants. In order to allow for a small number of inevaluable participants, the 
accrual ceiling will be set to 29 participants. If 3-4 participants per month enroll onto this study, 
accrual is expected to be completed in one year.
11 COLLABORATIVE AGREEMENTS
11.1
COLLABORATIVE RESEARCH AND DEVELOPMENT AGREEMENT (CRADA)  
The PROSTVAC vaccines will be provided under a Collaborative Research and Development 
Agreement (CRADA) with Bavarian Nordic, Inc., CRADA# 02377.
11.2
CLINICAL TRIALS AGREEMENT (CTA)
Bristol-Myers Squibb will be providing the study agent, nivolumab through a Clinical Trials 
Agreement (CTA), CTA# 1034-17.

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11.3
MATERIALS TRANSFER AGREEMENT (MTA)
Samples will be sent to the following collaborators through a Materials Transfer Agreement 
(MTA): General Electric (section 5.1.6.1); PerkinElmer (section 5.1.6.1); Definiens (section 
5.1.6.2); HTG Molecular Diagnostics (section 5.1.6.3.1); NantOmics (section 5.1.6.3.2); and 
Epic Sciences (section 5.1.7).
12 HUMAN SUBJECTS PROTECTIONS 
12.1
RATIONALE FOR SUBJECT SELECTION
Subjects treated on this study, will be men with castration resistant cancer (lead-in cohort) and 
localized adenocarcinoma of the prostate (neoadjuvant cohort).
Eligibility assessment will be based solely on the participant’s medical status.  Recruitment of 
participants onto this study will be through standard CCR mechanisms. No special recruitment 
efforts will be conducted.
12.2
INCLUSION OF WOMEN AND MINORITIES
Men of all races and ethnic groups are eligible for this trial. Women are excluded as prostate 
cancer does not exist in this population.
12.3
PARTICIPATION OF CHILDREN
Men under the age of 18 will not be eligible for participation in this study based on the fact that 
participants under 18 are unlikely to have this disease and there are unknown toxicities in 
pediatric participants.
12.4
PARTICIPATION OF SUBJECTS UNABLE TO GIVE CONSENT
Adults unable to give consent are excluded from enrolling in the protocol.  However, re-consent 
may be necessary and there is a possibility, though unlikely, that subjects enrolled in the protocol 
may permanently lose capacity to consent for themselves during the course of this study. For this 
reason and because there is a prospect of direct benefit from research participation (section 12.6), 
all subjects ≥ age 18 will be offered the opportunity to fill in their wishes for research and care, 
and assign a substitute decision maker on the “NIH Advance Directive for Health Care and 
Medical Research Participation” form so that another person can make decisions about their 
medical care in the event that they become incapacitated or cognitively impaired during the 
course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment 
Team (ACAT) for evaluation to assess ongoing capacity of the subjects and to identify an LAR 
as needed. Please see section 12.7.1 for consent procedure.
12.5
EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS
Potential risks of vaccine in this participant population include the range of side effects outlined 
in Section 14. PROSTVAC has been well tolerated in previous large trials.

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12.5.1 Alternative Approaches or Treatments
Participants will be advised verbally and in writing regarding the risks and benefits of this trial, 
treatment requirements, and alternative approaches to entering the trial. Written consents will be 
obtained.
12.5.2 Procedures to Eliminate or Minimize Potential Risks
This study may involve unforeseeable risks for participants, such as side effects whose exact 
nature and severity are unpredictable. Scrupulous care will be taken to minimize such side 
effects. All participants will be given blood tests, physical examinations, and scans, as described 
in the monitoring schedule, and must have a local physician to provide long-term care and 
monitoring for complications. Immediate medical treatment is available at the Clinical Center, 
NCI, Bethesda, Maryland, for any participants who suffer physical injury as a result of 
participation in this study. No compensation is available, but any injury will be evaluated and 
treated in keeping with the benefits or care to which participants are entitled under applicable 
regulations.
12.5.3 Provisions for Monitoring Data Collection to Ensure Subject Safety 
As information is gathered from this trial, clinical results will be shared with participants.  
Laboratory and clinical data will be frequently gathered and any new significant finding(s) found 
during the course of the research, which may affect a participant’s willingness to participate 
further, will be explained.
Confidentiality of information concerning participants will be maintained, including in all 
publications and presentations resulting from this study.  Names of participants or material 
identifying participants will not be released without permission, except as such release is 
required by law.  Records at the National Cancer Institute are maintained according to current 
legal requirements, and are made available for review, as required by the Food and Drug 
Administration or other authorized users, only under the guidelines established by the Federal 
Privacy Act.
12.5.4 Risks due to biopsy
All care will be taken to minimize risks that may be incurred by tumor sampling. However, there 
are procedure-related risks (such as bleeding, infection and visceral injury) that will be explained 
fully during informed consent. If participants suffer any physical injury as a result of the 
biopsies, immediate medical treatment is available at the NCI’s Clinical Center in Bethesda, 
Maryland.  Although no compensation is available, any injury will be fully evaluated and treated 
in keeping with the benefits or care to which participants are entitled under applicable 
regulations.
12.5.5 Research Blood Collection Risks
Risks of blood draws include pain and bruising in the area where the needle is placed, 
lightheadedness, and rarely, fainting. When large amounts of blood are collected, low red blood 
cell count (anemia) can develop. 

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12.5.6 Risks due to Radiation
The study will involve radiation from the following sources for CRPC cohort:

Up to 5 CT scans (C/A/P) per year for disease assessment not including one performed 
on separate study at screening

Up to 5 Tc99 scans per year for disease assessment not including one performed on 
separate study at screening

1 CT-guided biopsy 
Subjects in this study may be exposed up to 8.25 rem maximum annually.
The CT and bone scans that participant get in this study will expose them to the roughly the same 
amount of radiation up to 27.5 years of background radiation depending upon how much they 
weigh. The risk of getting cancer from the radiation exposure in this study is 0.8 out of 100 
(0.8%) and of getting a fatal cancer is 0.4 out of 100 (0.4 %). 
The study will have no radiation exposure for Neoadjuvant cohort:
12.5.7 Risks due to contrast dye (for CT and/or MRI)
If contrast dye is used, there is a risk for allergic reaction to the dye. Participants who are allergic 
to or sensitive to medications, contrast dye, iodine, or shellfish should notify their physician. 
Participants with kidney failure or other kidney problems should notify their physician. 
Participants may receive a contrast agent such as Gadolinium as part of your CT scan or MRI. 
Risks due to gadolinium are explained in consent document.
12.5.8 Risks due to MRI
Risks due to MRI are explained in the consent document.
12.6
RISKS/BENEFITS ANALYSIS
This study involves clinical research with an experimental vaccine designed to generate an 
immune response against antigens found in prostate cancer. Participants will undergo multiple 
vaccinations.  Side effects of the vaccine are outlined elsewhere.  Whether the vaccine will have 
any clinical effect is unknown; therefore, benefit cannot be promised from vaccine in addition to 
standard of care, nor the chance of benefit accurately predicted.  
The aim of this study is to see if PROSTVAC vaccine in combination with nivolumab is safe for 
castration resistant prostate cancer (CRPC) participants and to evaluate changes in the immune 
cells in the tumor after treatment in all other cancer participants.  The study drugs may help to 
control the disease.  There is a possibility of benefit for participants in this study, though it can’t 
be certain.  
Participation in this study is voluntary, and refusal will not result in penalty or loss of benefit to 
which the participant is otherwise entitled.   
Participation may be discontinued at any time without penalty, and the participant will be 
encouraged to discuss any concerns or questions.

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12.7
CONSENT PROCESS AND DOCUMENTATION
The informed consent document will be provided as a physical or electronic document to the 
participant or consent designee(s) (legally authorized representative [LAR] if participant is an 
adult unable to consent) for review prior to consenting.  A designated study investigator will 
carefully explain the procedures and tests involved in this study, and the associated risks, 
discomforts and benefits. In order to minimize potential coercion, as much time as is needed to 
review the document will be given, including an opportunity to discuss it with friends, family 
members and/or other advisors, and to ask questions of any designated study investigator. A 
signed informed consent document will be obtained prior to entry onto the study.
The initial consent process as well as re-consent, when required, may take place in person or 
remotely (e.g., via telephone or other NIH approved remote platforms used in compliance with 
local policy including HRPP Policy 303) per discretion of the designated study investigator and 
with the agreement of the participant/consent designee(s).  Whether in person or remote, the 
privacy of the subject will be maintained. Consenting investigators (and participant/consent 
designee, when in person) will be located in a private area (e.g., clinic consult room). When 
consent is conducted remotely, the participant/consent designee will be informed of the private 
nature of the discussion and will be encouraged to relocate to a more private setting if needed.  
Consent will be documented with required signatures on the physical document (which includes 
the printout of an electronic document sent to participant) or as described below, with a manual 
(non-electronic) signature on the electronic document. When required, witness signature will be 
obtained similarly as described for the investigator and participant.
Manual (non-electronic) signature on electronic document:
When a manual signature on an electronic document is used for the documentation of consent at 
the NIH Clinical Center, this study will use the following to obtain the required signatures:

Adobe platform (which is not 21 CFR Part 11 compliant) OR

iMedConsent platform (which is 21 CFR Part 11 compliant)
During the consent process, participants and investigators will view individual copies of the 
approved consent document on screens at their respective locations (if remote consent); the same 
screen may be used when in the same location, but is not required.
Both the investigator and the subject will sign the document using a finger, stylus or mouse. 
Note: Refer to the CCR SOP PM-2, Obtaining and Documenting the Informed Consent Process 
for additional information (e.g., verification of participant identity when obtaining consent 
remotely) found at: 
https://ccrod.cancer.gov/confluence/pages/viewpage.action?pageId=73203825. 
12.7.1 Consent Process for Adults Who Lack Capacity to Consent to Research Participation
For participants addressed in section 12.4, an LAR will be identified consistent with Policy 403 
and informed consent obtained from the LAR, as described in Section 12.7. 

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13 REGULATORY AND OPERATIONAL CONSIDERATIONS 
13.1
STUDY DISCONTINUATION AND CLOSURE
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to study participants, the 
Investigational New Drug (IND) sponsor and regulatory authorities.  If the study is prematurely 
terminated or suspended, the Principal Investigator (PI) will promptly inform study participants, 
the Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the 
termination or suspension.  Study participants will be contacted, as applicable, and be informed 
of changes to study visit schedule.
 Circumstances that may warrant termination or suspension include, but are not limited to:

Determination of unexpected, significant, or unacceptable risk to participants

Demonstration of efficacy that would warrant stopping   

Insufficient compliance to protocol requirements

Data that are not sufficiently complete and/or evaluable

Determination that the primary endpoint has been met

Determination of futility
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IRB and as applicable, Food and Drug Administration (FDA).
13.2
QUALITY ASSURANCE AND QUALITY CONTROL
Each clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion.  An individualized quality management 
plan will be developed to describe a site’s quality management.
Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution.
Following written Standard Operating Procedures (SOPs), the monitors will verify that the 
clinical trial is conducted and data are generated and biological specimens are collected, 
documented (recorded), and reported in compliance with the protocol, International Conference 
on Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements 
(e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). 
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local 
and regulatory authorities.
13.3
CONFLICT OF INTEREST POLICY
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical.  Therefore, any actual conflict of interest of persons who 
have a role in the design, conduct, analysis, publication, or any aspect of this trial will be 
disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be 

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required to have such conflicts managed in a way that is appropriate to their participation in the 
design and conduct of this trial.  The study leadership in conjunction with the National Cancer 
Institute has established policies and procedures for all study group members to disclose all 
conflicts of interest and will establish a mechanism for the management of all reported dualities 
of interest.
13.4
CONFIDENTIALITY AND PRIVACY
Participant confidentiality and privacy is strictly held in trust by the participating investigators, 
their staff, and the sponsor(s). This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participants. 
Therefore, the study protocol, documentation, data, and all other information generated will be 
held in strict confidence. No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor. 
All research activities will be conducted in as private a setting as possible.
The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and 
records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the participants in this study. The 
clinical study site will permit access to such records.
The study participant’s contact information will be securely stored at the clinical site for internal 
use during the study. At the end of the study, all records will continue to be kept in a secure 
location for as long a period as dictated by the reviewing IRB, Institutional policies, or sponsor 
requirements.
Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be stored at the NCI CCR. This will not include the participant’s contact or 
identifying information. Rather, individual participants and their research data will be identified 
by a unique study identification number. The study data entry and study management systems 
used by the clinical site and by NCI CCR research staff will be secured and password protected. 
At the end of the study, all study databases will be archived at the NCI CCR.
To further protect the privacy of study participants, a Certificate of Confidentiality has been 
issued by the National Institutes of Health (NIH).  This certificate protects identifiable research 
information from forced disclosure. It allows the investigator and others who have access to 
research records to refuse to disclose identifying information on research participation in any 
civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or 
local level. By protecting researchers and institutions from being compelled to disclose 
information that would identify research participants, Certificates of Confidentiality help achieve 
the research objectives and promote participation in studies by helping assure confidentiality and 
privacy to participants.
14 PHARMACEUTICAL INFORMATION  
14.1
RECOMBINANT FOWLPOX-PSA(L155)TRICOM  
Other Names: PROSTVAC-F/TRICOM; PROSTVAC-F

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Classification:  Recombinant fowlpox virus vector vaccine of the genus Avipoxvirus.
Product Description:  Recombinant Fowlpox-PSA(L155)/TRICOM is a recombinant 
fowlpox virus vector vaccine containing the genes for human PSA and three co-stimulatory 
molecules (designated TRICOM): B7.1, ICAM-1 (intercellular adhesion molecule-1), and 
LFA-3 (leukocyte function-associated antigen-3). The PSA gene coding sequence is modified to 
code for a single amino acid substitution [isoleucine to leucine at amino acid position 155 of the 
PSA antigen (designated L155)], which is designed to enhance immunogenicity. This modification 
occurs in a 10-mer, HLA-A2-restricted, immunodominant epitope of the antigen [designated PSA-3 
(amino acids 154-163)]. An attenuated, live, plaque-purified isolate from the POXVAC-TC strain 
of fowlpox virus was used as the parental virus for this recombinant vaccine. A plasmid vector 
containing the modified PSA gene and the genes for the three co-stimulatory molecules is used 
for in vivo recombination between the plasmid vector and parental fowlpox virus genome. The 
recombinant vaccine is manufactured by infection of primary chicken embryo dermal (CED) 
cells with the recombinant fowlpox virus. Fowlpox virus can infect mammalian cells and express 
the inserted transgenes to stimulate both humoral and cellular immunity, but cannot replicate in 
non-avian species, making systemic infections unlikely. 
How Supplied: Recombinant Fowlpox-PSA(L155)/TRICOM is supplied by Bavarian Nordic, 
Inc. in vials containing 0.75 mL of the vaccine at a final viral concentration titer of 2 x 109 
infectious units/mL formulated in phosphate-buffered saline containing 10% glycerol.
Preparation: Thaw vials completely at room temperature. Ensure the thawed contents are at the 
bottom of the upright vial and vortex vigorously at high power for at least ten seconds prior to 
dose preparation. Withdraw 0.5 mL (1 x 109 infectious units) into a 1 mL syringe for 
administration by subcutaneous injection.
Storage: Store intact vials of Recombinant Fowlpox-PSA(L155)/TRICOM at –70oC or colder. 
Stability: Shelf-life stability studies of the intact vials are ongoing. Once the intact vials are thawed, 
the vaccines maintain their potency for up to 4 days when stored at 2-8oC. Do not re-freeze thawed 
vials. Vials of Recombinant Fowlpox-PSA(L155)/TRICOM are for single-use only and do not 
contain a preservative. Administer prepared doses as soon as possible following preparation (i.e., 
within one hour). If necessary, store prepared doses at 2-8oC for up to 4 hours following 
preparation.
Route of Administration: Recombinant Fowlpox-PSA(L155)/TRICOM is administered by 
subcutaneous injection. 
Special Handling: Fowlpox virus is classified as a Biosafety Level 1 agent.  These agents are 
not known to cause disease in healthy human adults and are of minimal potential hazard to 
personnel and the environment under ordinary conditions of use. Clinicians can use techniques 
generally acceptable for nonpathogenic material. The recombinant vaccine is a preparation of a 
live virus (infectious for birds) containing DNA sequences derived from the human genome. 
Handle the recombinant vaccine as a hazardous biological substance and dispose of waste 
materials as hazardous biological waste, with incineration according to local institutional policy 

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and according to local, state, and federal regulations. Healthcare workers handling the 
recombinant fowlpox vaccine should avoid direct contact with pet birds for at least 72 hours after 
working with the agent. 
Preparation, Handling and Disposal Recommendations 

Strictly adhere to standard microbiological practices and techniques.

Limit/restrict access to preparation areas while dose preparation is in progress. 

Use appropriate infection control measures (e.g., thorough hand washing) after handling any 
materials.

Institute and follow policies for safe handling of sharps.

Perform all dose preparations in a certified Class II biological safety cabinet, generally using 
procedures, guidelines and personal protective apparel used during preparation of 
antineoplastic agents [e.g., minimizing creation of aerosols; no eating, drinking, handling 
contact lenses or applying cosmetics in the work area; using appropriate personal protective 
apparel - gowns, sterile latex gloves (double-gloving is recommended), respirator masks, 
protective eye wear, hair cover]. 

Decontaminate the biological safety cabinet prior to dose preparation with sterile gauze 
soaked in 10% bleach solution (0.52% sodium hypochlorite solution), or other appropriate 
disinfectant suitable for decontamination, rinsing, then wiping down with sterile gauze 
soaked in 70% alcohol. Consult specific manufacturer’s recommendations with respect to 
disinfectant concentration, contact time and method of application. 

Have all necessary supplies on-hand before beginning the preparation procedure. Develop a 
detailed worksheet outlining all supplies, dose calculations, and preparation procedures, and 
keep it available.

Place an empty biohazard sharps container lined with a leak-proof biohazard bag in or near 
the biosafety cabinet to dispose of all waste generated.

Transport the agent from the freezer to the work area in leak proof bag. 

Wipe or spray items used for dose preparation with 70% alcohol before placing in the 
biological safety cabinet. Disinfectants should remain in contact with the surfaces for at least 
five minutes prior to dose preparation. Avoid exposing the virus to disinfectants.

Wipe the syringe containing the prepared dose with 70% alcohol before removing it from the 
biological safety cabinet; transport it in a leak proof bag or container labeled with a 
biohazard symbol.

Place all waste into the sharps container lined with the leak proof biohazard bag and 
decontaminate the biological safety cabinet again by wiping down all surfaces with sterile 
gauze soaked in 10% bleach solution, or other appropriate disinfectant, rinsing, then wiping 
down with sterile gauze soaked in 70% alcohol. Following decontamination, dispose of 
personal protective apparel in the biohazard safety bag.

Incinerate waste according to institutional policy and according to local, state, and federal 
regulations.

Handle accidental spills similarly to antineoplastic spills and/or according to institutional 
policy:
o
Prevent others from entering the area and allow aerosols time to settle (approximately 10 
minutes). 
o
Use protective apparel, eyewear, mask, and gloves. 

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o
Cover spills with disposable absorbent towels. 
o
Decontaminate the area with 10% bleach solution, or other appropriate disinfectant 
suitable for decontamination, allowing approximately a 20-minute contact time. 
o
Dispose of all waste as biohazardous waste and incinerate according to institutional 
policy and according to local, state, and federal regulations. 
For more information about biohazard risk group classification and biohazard safety 
levels see:
A. Biosafety in Microbiological and Biomedical Laboratories; U. S. Department of Health 
and Human Services Centers for Disease Control and Prevention and National Institutes 
of Health. See current edition at:
http://www.cdc.gov/biosafety/publications/index.htm 
Patient Care Implications and Contraindications 
Cover vaccination sites with a sterile dry dressing (e.g., Telfa pad). Once the injection site is 
healed, no further barrier is necessary. As a precaution, protect injection sites that are exhibiting 
evidence of weeping, oozing or ulceration with a sterile dry dressing. In these circumstances, 
instruct patients to avoid direct contact of the injection site with susceptible individuals (e.g.; 
those who may be immunocompromised by disease or therapy). Instruct patients to avoid 
fathering a child for at least 4 months following therapy completion with the recombinant 
vaccine. Instruct patients receiving fowlpox vaccines to avoid direct contact with pet birds for at 
least 72 hours after vaccination or while there are any visible lesions at the injection site.
Due to the method of manufacturing (virus grown in primary chicken embryo dermal 
cells), patients with a history of allergy to eggs or egg products should not receive the 
vaccine.
14.2
RECOMBINANT VACCINIA-PSA(L155)/TRICOM 
Other Names: PROSTVAC-V/TRICOM; PROSTVAC-V
Classification:  Recombinant vaccinia virus vector vaccine of the genus Orthopoxvirus.
Product Description: Recombinant Vaccinia-PSA(L155)/TRICOM is a recombinant vaccinia 
virus vector vaccine containing the genes for human PSA and three co-stimulatory molecules 
(designated TRICOM): B7.1, ICAM-1 (intercellular adhesion molecule-1), and LFA-3 
(leukocyte function-associated antigen-3). The PSA gene coding sequence is modified to code 
for a single amino acid substitution [isoleucine to leucine at amino acid position 155 of the PSA 
antigen (designated L155)], which is designed to enhance immunogenicity. This modification 
occurs in a 10-mer, HLA-A2-restricted, immunodominant epitope of the antigen [designated PSA-3 
(amino acids 154-163)]. An attenuated, live, derivative of the Wyeth (New York City Board of 
Health) strain of vaccinia virus is used as the parental virus for the recombinant vaccine. A 
plasmid vector containing the modified PSA gene and the genes for the three co-stimulatory 
molecules is used for in vivo recombination between the plasmid vector and parental vaccinia 
virus genome. The recombinant vaccine is manufactured by infection of primary chicken embryo 
dermal (CED) cells with the recombinant vaccinia virus. Vaccinia virus can infect mammalian 
cells and express the inserted transgenes, and is a potent immune stimulator, eliciting both a strong 

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humoral and cellular immune response. Vaccinia virus is replication competent in mammalian cells, 
making systemic infections possible.
How Supplied: Recombinant Vaccinia-PSA(L155)/TRICOM is supplied by Bavarian Nordic, 
Inc. in vials containing 0.75 mL of the vaccine at a final viral concentration titer of 4 x 108 
infectious units/mL formulated in phosphate-buffered saline containing 10% glycerol. 
Preparation: Thaw vials completely at room temperature. Ensure the thawed contents are at the 
bottom of the upright vial and vortex vigorously at high power for at least ten seconds prior to 
dose preparation. Withdraw 0.5 mL (2 x 108 infectious units) into a 1 mL syringe for 
administration by subcutaneous injection.
Storage: Store intact vials of Recombinant Vaccinia-PSA(L155)/TRICOM at –70oC or colder.
Stability: Shelf-life studies of the intact vials are ongoing. Once the intact vials are thawed, the 
vaccines maintain their potency for up to 4 days when stored at 2-8oC. Do not re-freeze thawed 
vials. Vials of Recombinant Vaccinia-PSA(L155)/TRICOM are for single-use only and do not 
contain a preservative. Administer prepared doses as soon as possible following preparation (i.e., 
within one hour). If necessary, store prepared doses at 2-8oC for up to 4 hours following 
preparation.
Route of Administration: Recombinant Vaccinia-PSA(L155)/TRICOM is administered by 
subcutaneous injection. 
Special Handling and Precautions: Vaccinia virus is classified as a Biosafety Level 2 agent. 
These agents are associated with human disease and are of moderate potential hazard to 
personnel and the environment. The recombinant vaccine is a preparation of a live virus affecting 
humans and contains DNA sequences derived from the human genome. Handle the recombinant 
vaccine as an infectious hazardous biological substance and dispose of waste materials as 
infectious hazardous biological waste, with incineration according to local institutional policies 
and according to local, state, and federal regulations. 
Preparation, Handling and Disposal Recommendations 

Prepare a biosafety manual which advises personnel of special hazards and specific 
instructions on practices and procedures.

Post warning hazard signs on access doors, identifying the agents, the biosafety level, the 
name and phone number of the Principal Investigator or other responsible person, and any 
special requirements for entry.

Establish policies and procedures allowing only personnel who are knowledgeable of the 
potential hazards and meet specific entry requirements (e.g., immunization) into agent 
preparation or storage areas.

Strictly adhere to standard microbiological practices and techniques.

Limit/restrict access to preparation areas while dose preparation is in progress.

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
Use appropriate infection control measures (e.g., thorough hand washing) after handling any 
materials.

Institute and follow policies for safe handling of sharps. Use only needle-lock syringes and 
needles for dose preparation. Use extreme caution to prevent autoinoculation. Do not bend, 
shear, or replace the needle guard from the syringe following use. Promptly place used 
needles and syringes in puncture-resistant containers for disposal.

Perform all dose preparations in a certified Class II biological safety cabinet, generally using 
procedures, guidelines and personal protective apparel used during preparation of 
antineoplastic agents [e.g., minimizing creation of aerosols; no eating, drinking, handling 
contact lenses or applying cosmetics in the work area; using appropriate personal protective 
apparel - gowns, sterile latex gloves (double-gloving is recommended), respirator masks, 
protective eyewear, hair cover]. 

Perform all procedures carefully to minimize aerosol creation.

Decontaminate the biological safety cabinet prior to dose preparation with sterile gauze 
soaked in 10% bleach solution (0.52% sodium hypochlorite solution), or other appropriate 
disinfectant suitable for decontamination, rinsing, then wiping down with sterile gauze 
soaked in 70% alcohol. Consult specific manufacturer’s recommendations with respect to 
disinfectant concentration, contact time and method of application. 

Have all necessary supplies on-hand before beginning the preparation procedure. Develop a 
detailed worksheet outlining all supplies, dose calculations, and preparation procedures, and 
keep it available.

Place an empty biohazard sharps container in the biosafety cabinet to dispose of all waste 
generated.

Transport the agent from the freezer to the work area in leak proof bag. 

Wipe or spray items used for dose preparation with 70% alcohol before placing in the 
biological safety cabinet. Disinfectants should remain in contact with the surfaces for at least 
five minutes prior to dose preparation. Avoid exposing the virus to disinfectants.

Wipe the syringe containing the prepared dose with 70% alcohol before removing it from the 
biological safety cabinet; transport it in a leak proof bag or container labeled with a 
biohazard symbol.

Place all waste into a sharps container lined with the leak proof biohazard bag and 
decontaminate the biological safety cabinet again by wiping down all surfaces with sterile 
gauze soaked in 10% bleach solution, or other appropriate disinfectant, rinsing, then wiping 
down with sterile gauze soaked in 70% alcohol. Following decontamination, dispose of 
personal protective apparel in the biohazard safety bag.

Place all waste and protective apparel in a leak proof biohazard bag, and place the bag inside 
a biohazard sharps container for incineration according to institutional policy and according 
to local, state, and federal regulations.

Handle accidental spills similarly to antineoplastic spills and/or according to institutional 
policy:
o
Prevent others from entering the area and allow aerosols time to settle (approximately 10 
minutes). 
o
Use protective apparel, eyewear, mask, and gloves. 
o
Cover spills with disposable absorbent towels. 

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o
Decontaminate the area with 10% bleach solution, or other appropriate disinfectant 
suitable for decontamination, allowing approximately a 20-minute contact time. 
o
Dispose of all waste and protective apparel as infectious biohazardous waste and 
incinerate according to institutional policy and according to local, state, and federal 
regulations. 
For more information about biohazard risk group classification and biohazard safety levels: 
B. Biosafety in Microbiological and Biomedical Laboratories; U. S. Department of 
Health and Human Services Centers for Disease Control and Prevention and 
National Institutes of Health. See current edition at: 
http://www.cdc.gov/biosafety/publications/index.htm 
Precautions for Healthcare Workers 
Recombinant vaccinia virus transmission risk to exposed healthcare workers is unknown. To 
date, no reports of transmission to healthcare personnel from vaccine recipients have been 
published. If appropriate infection control precautions are observed (such as covering the 
vaccination site and washing hands after contact with the vaccination site or bandages), 
healthcare workers are probably at less risk of infection than laboratory workers because of the 
smaller volume and lower titers of virus in clinical specimens as compared with laboratory 
material. However, because of the potential risk for transmission, healthcare personnel who 
prepare or administer doses of recombinant vaccinia vaccine or have direct contact with 
contaminated dressings or other infectious material from participants in clinical studies must 
adhere to appropriate infection control measures and should be offered vaccination with the 
licensed vaccinia vaccine. Do not administer routine, non-emergency vaccination with the 
licensed vaccinia vaccine to healthcare workers, if they, or for at least three weeks after 
vaccination, their close household contacts (close household contacts are those who share 
housing or have close physical contact): 
1. have active eczema or a history of eczema or atopic dermatitis, or Darier’s disease.
2. have other acute, chronic, or exfoliative skin conditions (e.g., burns, impetigo, varicella 
zoster, severe acne, or other open rashes or wounds), until the condition resolves.
3. are pregnant or intend to become pregnant within 4 weeks of vaccination or are breast-feeding.
4. are immunodeficient or immunocompromised (by disease or therapy), including HIV 
infection.
Additionally, do not administer routine, non-emergency vaccination with the licensed vaccinia 
vaccine to healthcare workers if the vaccinee:  

has a moderate or severe acute illness, until the illness resolves.

is less than 18 years of age, unless specifically indicated.

is undergoing topical steroid therapy for inflammatory eye diseases or undergoing therapy 
with systemic steroids; potential immune suppression increases risk for vaccinia-related 
complications. 

has a history of allergy or serious reaction to prior vaccinia vaccination or any of the 
vaccine’s components.

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
As a precaution, the CDC recommends that individuals with known cardiac disease (e.g., 
previous MI, angina, CHF, cardiomyopathy, stroke or TIA) or who have > 3 known risk 
factors for cardiac disease (e.g., hypertension, hypercholesterolemia, diabetes, first degree 
relative with onset of cardiac complications prior to age 50, smoker), not receive routine, 
non-emergency, prophylactic vaccination with the licensed vaccinia vaccine while a possible 
causal relationship between vaccination and cardiac events is being evaluated.
Avoid exposure to the recombinant vaccinia vaccine, any contaminated dressings, or other 
infectious materials from patients, or the patient’s inoculation site if you are pregnant or breast-
feeding; have a history or presence of active eczema or atopic dermatitis; have acute, chronic or 
exfoliative skin conditions; or, are immunocompromised. More information on vaccinia 
precautions for healthcare workers can be obtained from 
http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5010a1.htm#tab2 and 
http://www.cdc.gov/mmwr/PDF/rr/rr5207.pdf.
The CDC is the only source of the licensed vaccinia vaccine. The CDC will provide vaccinia 
vaccine to protect laboratory and other healthcare personnel, whose occupations place them at 
risk of exposure to vaccinia and other closely related orthopoxviruses, including vaccinia 
recombinants. The vaccine should be administered under the supervision of a physician selected 
by the study institution. Revaccination is recommended every 10 years. For instructions on 
obtaining the licensed vaccinia vaccine, contact Drug Services, National Center for Infectious 
Diseases, CDC at (404) 639-3670.
Recombinant Vaccinia Vaccine Patient Care Implications, Contraindications and Potential 
Complications 
Patient Care Implications and Contraindications
Cover vaccination sites with a sterile dry dressing (e.g., Telfa pad). Instruct patients on proper hand-
hygiene, sterile dressing care, bathing, laundering of clothing, etc. Treat patient bandages or 
dressings removed from the vaccination site as infectious waste and dispose in appropriate 
biohazard containers. Do not administer the recombinant vaccinia vaccine if the recipient, or for at 
least three weeks after vaccination, their close household contacts (close household contacts are 
those who share housing or have close physical contact): 

have active eczema or a history of eczema or atopic dermatitis, or Darier’s disease.

have other acute, chronic, or exfoliative skin conditions (e.g., burns, impetigo, varicella zoster, 
severe acne, contact dermatitis, psoriasis, herpes or other open rashes or wounds), until the 
condition resolves.

are pregnant or intend to become pregnant (due to the potential risk of fetal vaccinia); or are 
breast-feeding (due to the potential risk of contact transmission and inadvertent inoculation). 
It is currently unknown if vaccinia virus or antibodies are excreted in breast milk.  Patients 
(i.e., vaccinees) should avoid fathering a child for at least 4 months following completion of 
therapy with the recombinant vaccine.

are in close contact with children less than 3 years of age (due to the potential risk of contact 
transmission and inadvertent inoculation). 

are immunodeficient or immunocompromised (by disease or therapy), including individuals 
with HIV infection.

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Additionally, do not administer the recombinant vaccinia vaccine if the vaccinee: 
1. has a moderate or severe acute illness, until the illness resolves.
2. is undergoing topical steroid therapy for inflammatory eye diseases, or undergoing therapy 
with systemic steroids; potential immune suppression increases risk for vaccinia-related 
complications.
3. At this time, until a more definitive causal relationship is determined, it is recommended that 
patients with known CHF or clinically significant cardiomyopathy, not be vaccinated with 
recombinant vaccinia-based vaccines, due to the potential for development of myocarditis 
and/or pericarditis. 
Although the CDC believes that there is no evidence to conclude that the licensed vaccinia 
vaccine used in the Smallpox Vaccination Program causes angina or heart attacks, it 
acknowledges a possible causal relationship between the vaccination and heart inflammation.  
The CDC continues to study the relationship, but in the meantime, recommends excluding 
individuals with underlying heart disease from participation in the current Smallpox 
Vaccination Program. Patients are being immunized with recombinant vaccinia vaccines with 
therapeutic intent and will be evaluated for cardiovascular risk factors and recent or 
symptomatic cardiac events per protocol eligibility criteria. Patients will be encouraged to 
minimize cardiovascular disease risks and encouraged to follow risk reduction according to 
standard medical practice.   
Due to the method of manufacturing (virus grown in primary chicken embryo dermal cells), do 
not administer the recombinant vaccinia vaccine to patients with a history of allergy to eggs or 
egg products. Do not administer the recombinant vaccinia vaccine to patients with a history of 
allergy or serious reaction to prior vaccinia vaccination (e.g., smallpox vaccination).
Potential Complications Associated With Recombinant Vaccinia Vaccination 
When vaccinia vaccine is administered by scarification for vaccination against smallpox, 
expected local reactions in individuals that have not previously been vaccinated with vaccinia 
include the appearance of a red papule in 3-4 days, followed by vesiculation in 5-6 days, and 
then the formation of a pustule on days 8-9. A large area of erythema may surround the vesicle 
and pustule. A crusted scab usually forms by the second week and sloughs by the third week, 
leaving a well-formed scar. Maximal viral shedding occurs from days 4-14, but can continue 
until the scab is shed from the skin. Other normal local reactions can include development of 
local satellite lesions, regional lymphadenopathy that can persist for weeks following healing of 
the skin lesion, considerable local edema, and intense inflammation from the vaccination (i.e., 
viral cellulites), which may be confused with bacterial cellulites. Systemic symptoms typically 
occur between 8-10 days post-vaccination and include fever, malaise, headache, chills, nausea, 
myalgia, local lymphadenopathy, soreness and intense erythema surrounding the vaccination 
site.
When administered by scarification in individuals that have previously been vaccinated with 
vaccinia, expected local reactions include the appearance of a clear cut pustule 6-8 days 
following vaccination or the development of an area of definite induration around a central lesion 
that may be an ulcer or scab 6-8 days following vaccination. The response to re-vaccination 
depends on the degree of residual immunity following previous vaccination. Similar systemic 
symptoms may occur, but typically at a lower frequency. 

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When recombinant vaccinia vaccines are administered by intradermal, intralesional, 
subcutaneous or intramuscular routes of injection, milder local reactions are expected, but similar 
systemic symptoms may occur.
There have been a number of complications reported in the literature associated with vaccinia 
vaccination for smallpox. Reported complications from vaccinia vaccine when given by 
scarification include: a) auto-inoculation of other sites with vaccinia, b) generalized vaccinia, c) 
eczema vaccinatum, d) progressive vaccinia (vaccinia necrosum), or e) post-vaccinial 
encephalitis.  In a 1968 ten-state survey, cases of these complications per million vaccinations in 
adult recipients (> 20 years of age) of vaccinia primary vaccination and revaccination were:
Primary Vaccination
Revaccination
auto-inoculation
606.1
25
generalized vaccinia
212.1
9.1
eczema vaccinatum
30.3
4.5
progressive vaccinia
none reported
6.8
postvaccinial encephalitis
none reported
4.5
Based on a 1968 national survey, the number of deaths in primary vaccinees was approximately 
1 per million and the number of deaths in recipients of revaccination was approximately 0.25 per 
million. Deaths were most often the result of postvaccinial encephalitis or progressive vaccinia.
Information has been reported by the US Department of Defense (DoD) during the post-
vaccination surveillance assessment of adverse events in military personnel following 
implementation of a Smallpox Vaccination Program from the period of December 13, 2002 
through May 28, 2003. Although not directly comparable to historical numbers, due to 
differences in multiple population variables, estimated cases (number of cases per million 
vaccinations based on vaccination of 450,293 personnel, with a median age of 26 years and 
70.5% as primary vaccinees) of these same complications per million vaccinations were:
auto-inoculation
107
generalized vaccinia
80
eczema vaccinatum
none reported
progressive vaccinia
none reported
postvaccinial encephalitis
2.2
 

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Generally, self-limited adverse reactions that can be serious, but not life-threatening include 
autoinoculation, erythematous and urticarial rashes, and generalized vaccinia. More serious life-
threatening complications include progressive vaccinia, eczema vaccinatum, and post-vaccinial 
encephalitis/encephalomyelitis. The complications of vaccinia vaccination may involve a number 
of different reactions:

Non-specific erythematous or urticarial rashes: These rashes can appear 
approximately 10 days after vaccination and may sometimes be confused with 
generalized vaccinia but are generally self-limiting. Patients are usually afebrile and these 
benign rashes usually resolve spontaneously within 2-4 days. Erythema multiforme can 
present as different types of lesions, including macules, papules, urticaria, and bull’s eye 
lesions (dark papule or vesicle surrounded by a pale zone and an area of erythema). These 
lesions may be extremely pruritic, lasting up to four weeks. Rarely, more serious bullous 
erythema multiforme (Stevens-Johnson syndrome) may occur, requiring hospitalization. 
Vaccinia Immune Globulin (VIG) therapy is not indicated to treat these rashes. 
Supportive care measures are warranted since these rashes are likely manifestations of an 
immune response or hypersensitivity reaction to the vaccine and are not likely to contain 
vaccinia virus.

Bacterial Infection: Vaccination site infection, most likely due to staphylococcus and 
streptococcus normal skin flora, is rare. Onset is approximately 5 days post-vaccination 
and is more common in children. Appropriate antibiotic therapy is required.

Inadvertent Inoculation: This can occur in the vaccinee (autoinoculation) as well as in 
close contacts (contact transmission). Accidental infection is the most common 
complication of vaccinia vaccination, accounting for approximately 50% of all 
complications associated with vaccination and revaccination. This usually results from 
autoinoculation of vaccinia virus transferred from the site of the vaccination. Sites 
typically involved include the face, eyelids, nose, mouth, genitalia, or rectum, but can 
also involve the arms, legs, and trunk. Contact transmission of vaccinia, with 
accompanying toxicities, may occur when a recently vaccinated individual has contact 
with a susceptible individual.  In a 1968 ten-state survey, contact transmissions were 
reported to occur at a rate of 27 infections per million vaccinations.  The age group in 
which contact transmission occurred most commonly was in children  5 years. Eczema 
vaccinatum as a result of contact transmission may result in a more severe syndrome than 
that seen in vaccinees, perhaps because of multiple simultaneous inoculations. About 
30% of eczema vaccinatum cases reported in the 1968 ten-state survey were a result of 
contact transmission. It is possible that the number of cases of contact transmission would 
be greater in today’s population, due to a largely unvaccinated patient population against 
smallpox. Contact transmission rarely results in postvaccinial encephalitis or progressive 
vaccinia. Most cases of inadvertent inoculation usually resolve without specific therapy 
and resolution of lesions follow the same course as the vaccination site in 
immunocompetent individuals. VIG can be used for severe cases involving extensive 
lesions or if comorbid conditions exist. VIG is contraindicated in the presence of isolated 
keratitis due to the risk of increased corneal scarring. VIG use can be considered in cases 
of ocular implantation, with keratitis, if vision-threatening or if other life-threatening 
vaccinia-related complications exist that require VIG therapy.  

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
Generalized vaccinia: Generalized vaccinia (GV) is characterized by a disseminated 
maculopapular or vesicular rash of varying extent on any part of the body and typically 
develops 6-9 days after vaccination. The lesions follow the same course as the 
vaccination site lesion. The lesions are hematogenously spread and may contain vaccinia 
virus. In immunocompetent individuals, the rash is generally self-limiting and requires 
supportive care therapy. VIG treatment can be used in severe cases for patients who are 
systemically ill and whose condition might be toxic or who have serious underlying 
immunosuppressive illnesses.
The differential diagnosis of GV includes eczema vaccinatum, erythema multiforme, 
inadvertent inoculation at multiple sites, and uncommonly, early stages of progressive 
vaccinia or other vesicular diseases (e.g., severe chickenpox or disseminated herpes). 
Several publications have investigated the reporting of GV among those individuals who 
received smallpox vaccinations during 2003.  Out of 38,440 vaccine recipients, 29 reports 
of possible GV during January 2003–December 2003 were identified but only 2 reports 
met the case definition. More than 75% of the reports received a final diagnosis of 
hypersensitivity reaction or nonspecific rash after review by dermatologists or because 
laboratory results were negative for vaccinia and other orthopoxviruses.  Of 74 cases 
investigated in 753,226 smallpox vaccinations administered in December 2002 to 
December 2004, 50 (67.6%) met the case definition of possible GV.  Cases occurred 
more frequently in primary vaccinees (rate, 81 per 1 million vaccinees) than in those 
revaccinated (rate, 32 per 1 million vaccinees).  However, none met the case definition of 
probable or confirmed GV, including 15 with virologically negative laboratory 
evaluations (e.g., culture, polymerase chain reaction, or skin biopsy).  Twenty-one reports 
of postscab lesions were made between January and August 2003 among 37,542 
smallpox vaccinees.  The lesions (scab and/or fluid) of seven patients were tested for 
vaccinia virus by use of polymerase chain reaction and/or immunohistochemistry; all 
were found to be negative.  In addition, the postscab lesions of four of the patients were 
biopsied.  The results from two of the biopsies suggested an allergic contact dermatitis, 
and results of one each demonstrated chronic dermatitis and squamous cell carcinoma.  
None of the four biopsied lesions had histologic evidence of viropathic changes and no 
evidence supported smallpox vaccination as a cause for any of the lesions.  

Eczema vaccinatum: Eczema vaccinatum is a serious complication in persons with 
eczema and other types of chronic or exfoliative skin conditions. It can also occur among 
eczematous contacts of recently vaccinated persons. Vaccinial lesions (generalized 
papular, vesicular or pustular lesions) develop on areas of the skin that are, or had at one 
time been, eczematous. These areas become highly inflamed and lesions may spread to 
healthy skin. The rash is often accompanied by fever and individuals are systemically ill. 
The fatality rate for untreated cases (prior to availability of VIG) has been reported from 
30-40%. Following availability of VIG, mortality was reduced to approximately 7%. 
Early diagnosis and prompt treatment with VIG is necessary to reduce mortality. 

Progressive vaccinia: Progressive vaccinia is the most serious cutaneous complication, 
occurring when the local vaccination lesion fails to heal and develops progressive 
necrosis, with destruction of large areas of skin, subcutaneous tissue, and underlying 
structures. Progressive lesions may spread to other skin surfaces and to bone and viscera.   
Progressive vaccinia is associated with a high mortality rate. This complication has been 
seen in patients with a compromised immune system due to a congenital deficiency, 

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lymphoproliferative 
disease, 
immunosuppressive 
treatment, 
or 
HIV 
infection. 
Management should include aggressive VIG therapy.

Post-Vaccinial Encephalitis/Encephalomyelitis: Vaccinial complications affecting the 
CNS are unpredictable. Post-vaccinial encephalitis typically affects children < 2 years of 
age and is characterized by an onset of symptoms 6-10 days following vaccination, which 
include seizures, hemiplegia, aphasia, and transient amnesia. Histopathological changes 
include generalized cerebral edema, mild lymphocytic meningeal infiltration, ganglion 
degenerative changes and perivascular hemorrhages. Older children and adults can 
develop encephalitis or encephalomyelitis characterized by an onset of symptoms 11-15 
days following vaccination, which include fever, vomiting, headache, malaise, and 
anorexia, progressing to loss of consciousness, amnesia, confusion, disorientation, 
restlessness, delirium, drowsiness, seizures and coma. Histopathological changes include 
demyelination with lymphocytic infiltration, but limited cerebral edema. Mortality rates 
have ranged from 15-25%, with 25% of patients who recover being left with varying 
degrees and types of neurological deficits. VIG has not been shown to be effective in 
treating 
CNS 
disease 
and 
is 
not 
recommended. 
Post-vaccinial 
encephalitis/encephalomyelitis are diagnoses of exclusion and are not believed to be a 
result of replicating vaccinia virus.  Although no specific therapy exists, supportive care, 
anticonvulsants, and intensive care might be required. A review of vaccinia-related 
deaths (68) during a 9-year period (1959–1966 and 1968) revealed that among first-time 
vaccines, 36 (52%) patients died as a result of post-vaccinial encephalitis.    

Fetal Vaccinia: Fetal vaccinia is a rare, but serious complication following vaccinia 
vaccination during pregnancy or shortly before conception (e.g., within four weeks).  To 
date, fewer than 50 cases have been reported and often result in fetal or neonatal death. 
Efficacy of VIG therapy in a viable infant or used prophylactically in women during 
pregnancy is unknown. The CDC has established a National Smallpox Vaccine in 
Pregnancy Registry. This registry will follow women during their pregnancies and their 
babies, after they are born, to determine the outcome of such pregnancies. The CDC can 
be contacted at (404) 639-8253.

Myocarditis/Pericarditis:  The CDC has recommended a temporary medical deferral to 
the voluntary Smallpox Vaccination Program for persons with heart disease or 
cardiovascular risk factors (March 25, 2003) and issued “interim supplementary 
information” regarding evidence that smallpox vaccination may cause myocarditis and/or 
pericarditis (March 31, 2003) in people recently vaccinated with the smallpox vaccine. 
The cardiac events reported include myocardial infarction, angina, myocarditis, 
pericarditis, and myopericarditis. Although the CDC believes that there is no evidence to 
conclude that the licensed vaccinia vaccine causes angina or heart attacks, it 
acknowledges a possible causal relationship between the vaccination and heart 
inflammation.  The CDC continues to study the relationship, but in the meantime, 
recommends that individuals with underlying heart disease be excluded from 
participation in the current Smallpox Vaccination Program. While it is currently not 
possible to fully evaluate the risk of cardiac events or the risk of myocarditis, pericarditis, 
or myopericarditis associated with vaccinia vaccination, it is reasonable to inform 
patients participating in studies using recombinant vaccinia virus of these reports and 
provide relevant guidance for evaluating these events. Further investigation from the 
ongoing vaccine program may provide additional data regarding an association or lack of 

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association with cardiovascular disease. Patients are being immunized with recombinant 
vaccinia vaccines with therapeutic intent and will be evaluated for cardiovascular risk 
factors and recent or symptomatic cardiac events per protocol eligibility criteria. Patients 
will be encouraged to minimize cardiovascular disease risks and encouraged to follow 
risk reduction according to standard medical practice. At this time the evidence for an 
association with myocarditis, pericarditis, or myopericarditis seems plausible, but a rare 
event.  If not otherwise excluded, patients with known CHF or clinically significant 
cardiomyopathy requiring treatment should be excluded from protocol eligibility.      
Out of a total of 540,824 military personnel vaccinated with a New York City Board of 
Health strain of vaccinia from December 2002 through December 2003, 67 developed 
symptomatic myopericarditis.  In the 61 ECGs that were reviewed, an identifiable 
abnormality was evident in 46 (75.4%).  The most common abnormalities included ST-
segment changes observed evident in 40 patients (65.6%); 5 patients (8.2%) had normal 
variant early repolarization, and T-wave abnormalities were noted in 11 patients (18.0%).  
In addition, cardiac enzymes were elevated in 60 of 61 (98.4%) patients evaluated with 
this assay.  On follow-up of 64 patients, all patients had objective normalization of 
electrocardiography, echocardiography, graded exercise testing, laboratory testing, and 
functional status; 8 (13%) reported atypical, non-limiting persistent chest discomfort.  
Among 37,901 health care workers vaccinated with the identical strain, 21 
myo/pericarditis cases were identified; 18 (86%) were revacinees. Twelve met criteria for 
either myocarditis or myopericarditis, and 9 met criteria for pericarditis only (6 suspected 
and 3 probable).  The severity of myo/pericarditis was mild, with no fatalities, although 9 
patients (43%) were hospitalized.
14.3
NIVOLUMAB
Amino Acid Sequence:  4 polypeptide chains, which include 2 identical heavy chains with 440 
amino acids and 2 identical light chains.
Other Names:BMS-936558, MDX1106
Classification:Anti-PD-1MAb
M.W.:  146,221 daltons
Mode of Action:  Nivolumab targets the programmed death–1 (PD-1, cluster of differentiation 
279 [CD279]) cell surface membrane receptor. PD-1 is a negative regulatory receptor expressed 
by activated T and B lymphocytes. Binding of PD-1 to its ligands, programmed death–ligand 1 
(PD-L1) and 2 (PD-L2), results in the downregulation of lymphocyte activation. Nivolumab 
inhibits the binding of PD-1 to PD-L1 and PD-L2.  Inhibition of the interaction between PD-1 
and its ligands promotes immune responses and antigen-specific T-cell responses to both foreign 
antigens as well as self-antigens.
Description:  Nivolumab Injection is a clear to opalescent, colorless to pale yellow liquid; light 
(few) particulates may be present. The drug product is a sterile, nonpyrogenic, single-use, 
isotonic aqueous solution formulated in sodium citrate, sodium chloride, mannitol, 
diethylenetriamine pentacetic acid (pentetic acid) and polysorbate 80 (Tween® 80), pH 6.0.

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How Supplied:  Nivolumab will be supplied by Bristol-Myers Squibb as 100 mg vials (10 mg/mL) 
with a 0.7mL overfill.  It is supplied in 10 mL type I flint glass vials, with butyl rubber stoppers and 
aluminum seals.
Preparation:  Nivolumab injection can be infused undiluted (10 mg/mL) or diluted with 0.9% 
Sodium Chloride Injection, USP or 5% Dextrose, USP to concentrations no less than 0.35 
mg/mL. When the dose is fixed (e.g., 240 mg or 480 mg flat dose), nivolumab injection can be 
infused undiluted or diluted so as not to exceed a total infusion volume of 160 mL.
Storage: Vials of Nivolumab injection must be stored at 2°-8°C (36°-46°F) and protected from 
light, freezing and shaking. 
Stability:  Shelf-life surveillance of the intact vials is ongoing.
The administration of undiluted and diluted solutions of Nivolumab must be completed within 24 
hours of preparation.  If not used immediately, the infusion solution may be stored up to 24 hours 
in a refrigerator at 2°-8°C (36°-46°F) and a maximum of 8 hours of the total 24 hours can be at 
room temperature (20°-25°C, 68°-77°F) and room light.  The maximum 8-hour period under 
room temperature and room light conditions includes the product administration period.
Toxicity: The following related adverse events have been reported with nivolumab monotherapy 
at an incidence of  > 5%: fatigue, rash, pruritis, diarrhea, nausea, hypothyroidism, decreased 
appetite, and musculoskeletal pain. Immune-related adverse events have also been reported and 
may be severe or life-threating. 
Route of Administration:   Intravenous infusion.  Do not administer as an IV push or bolus 
injection.
Method of Administration:  Administer through a 0.2 micron to 1.2 micron pore size, low-
protein binding polyethersulfone membrane in-line filter. 
Potential Drug Interactions: No incompatibilities between Nivolumab injection and polyvinyl 
chloride (PVC), non-PVC/non-DEHP (di(2-ethylhexyl)phthalate) IV components, or glass 
bottles have been observed.
Destruction of nivolumab provided by BMS:
On-site destruction of unused vials of investigational nivolumab should be conducted according to 
institutional standards.  A copy of the drug destruction certificate must be maintained for provision 
to BMS at the end of the study.

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49.
Wolchok JD, Hoos A, O'Day S, Weber JS, Hamid O, Lebbe C, Maio M, Binder M, 
Bohnsack O, Nichol G et al: Guidelines for the evaluation of immune therapy activity 
in solid tumors: immune-related response criteria. Clin Cancer Res 2009, 
15(23):7412-7420.

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16 APPENDICES
16.1
APPENDIX A: PERFORMANCE STATUS CRITERIA
ECOG Performance Status Scale
Karnofsky Performance Scale
Grade
Descriptions
Percent
Description
100
Normal, no complaints, no 
evidence of disease.
0
Normal activity.  Fully active, able to carry on all 
pre-disease performance without restriction.
90
Able to carry on normal 
activity; minor signs or 
symptoms of disease.
80
Normal activity with effort; 
some signs or symptoms of 
disease.
1
Symptoms, but ambulatory.  Restricted in physically 
strenuous activity, but ambulatory and able to carry 
out work of a light or sedentary nature (e.g., light 
housework, office work).
70
Cares for self, unable to carry 
on normal activity or to do 
active work.
60
Requires occasional 
assistance, but is able to care 
for most of his/her needs.
2
In bed <50% of the time.  Ambulatory and capable of 
all self-care, but unable to carry out any work 
activities.  Up and about more than 50% of waking 
hours.
50
Requires considerable 
assistance and frequent 
medical care.
40
Disabled, requires special 
care and assistance.
3
In bed >50% of the time.  Capable of only limited 
self-care, confined to bed or chair more than 50% of 
waking hours.
30
Severely disabled, 
hospitalization indicated.  
Death not imminent.
20
Very sick, hospitalization 
indicated. Death not 
imminent.
4
100% bedridden.  Completely disabled.  Cannot 
carry on any self-care.  Totally confined to bed or 
chair.
10
Moribund, fatal processes 
progressing rapidly.
5
Dead.
0
Dead.

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16.2
APPENDIX B: VACCINIA-PROSTVAC PATIENT INSTRUCTION SHEET
1.
What vaccination site reactions can you expect?
2.
How should you care for the vaccination site? 
3.
Are there any activities I should avoid?
4.
What about contact with other people?
5.
Who do I contact when I have a question?
 1.
What vaccination site reactions can you expect?
A typical reaction in a patient who has been previously vaccinated with vaccinia includes the 
appearance of a small swelling in 2-3 days that may enlarge to 1-2 inches across, a small blister or 
pustule after 5-7 days, and healing with little scarring within 2-3 weeks. Some patients may have 
very little skin reaction.   Often the leg that received the vaccine may become swollen.  Swollen or 
tender lymph nodes (“glands”) in the groin may be felt. A fever to 100-101F may occur on the 
second or third day.  You may notice that you feel tired for 3 or 4 days. The vaccination site may 
itch for about 2 weeks while the scab is forming.  You can take acetaminophen ("Tylenol") if you 
have any aches or fever but you should avoid aspirin.  If fever continues for more than a day or 
two, you should call to speak to the clinic nurse or the research nurse. 
In patients who have never received vaccinia or in some who received it a very long time ago, a 
red swelling is followed by a blisters on day 5 to 6 and then the formation of a pustule (or "boil") 
1-2 inches in diameter on day 9 to 11.  A large area of redness may surround this area.  A crusted 
scab usually forms by the second week and falls off by the third week leaving a scar roughly 1/2 
inch in diameter.  Fever and malaise (the "blahs") may occur during the blister and pustular phases. 
Swollen and tender lymph nodes may persist for months. Many of the local toxicities described 
(e.g., pustule and scab formation) are typical of reactions seen when vaccinia is administered via 
scarification or intradermal administration. These reactions may be seen, but are usually not seen 
when administered via subcutaneous injection.
 2.
How should you care for the vaccination site?
Live vaccinia virus is in skin cells at the vaccination site during the 1-2 weeks until healing has 
occurred.  Maximal viral "shedding" from the vaccination site occurs from days 4-14, but can 
continue until the scab falls off from the skin. After that there is no vaccinia virus in your body.  
You can spread the virus to other parts of your body or to other people by touching the vaccination 
site and then touching your eyes, mouth, a cut or some other break in the skin.  You do not pass 
vaccinia virus by coughing or sneezing or by sharing food or cups and dishes.

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In general, frequent careful hand washing by you and by any persons in physical contact with you 
is the best way to prevent transmission of virus.  You should also use two types of barriers over 
your vaccination site at all times until the scab is gone.  These barriers are (1) the bandage and (2) 
clothing (pants or elbow length sleeves depending on the site of vaccination).  These barriers 
should remain in place until the scab has fallen off.
For dressing care you will have a bag with some no-stick "First-Aid" or "Telfa" pads, disposable 
gloves, and zip-lock plastic bags.  If you run out of supplies between visits, you can use a dry 
sterile bandage (gauze or "First-Aid" or "Telfa") from the drug store.
The no-stick pad ("First-Aid" or "Telfa" pad) dressing should be worn until the site has healed.  If 
it remains clean and dry and is not coming off, you do not need to change it.  If the dressing gets 
wet either from drainage from the vaccination or from water when you are showering or if it starts 
coming off, you should remove it and put on a clean bandage.  Wear the gloves when handling the 
old dressings.  Put the old dressing and the gloves in the zip-lock bag, then wash your hands, put 
on the new bandage, and wash your hands again.  You do not need to wear gloves for the new 
bandage.  You do not need to wash the vaccination site, but while the dressing is off, you may 
wash it lightly with a cloth, soap, and water.  If you do wash, blot the site dry with a towel (don't 
rub), then put the wash cloth and the towel in the laundry.  Do not let the shower run on the non-
bandaged site because live virus could be washed onto other areas of your body. Do not put any 
steroid cream, medicated creams, or other ointments on the vaccination site.
Before you throw away the zip-lock bag with the old dressing and gloves in it, pour a little bleach 
(about a quarter cup) in the bag to help kill any virus.
Wash your hands after each step.
 3. Are there any activities I should avoid or take special care?
You should not go swimming until the vaccination site has healed and you no longer need to wear 
a bandage on it. 
If you wear contact lenses, have removable dentures, have a colostomy or any other "open" area on 
your body that needs daily care, always wash your hands very well before handling your contact 
lenses, dentures, dressings, etc.  Take care of all of these procedures before changing your 
vaccination dressing.
4.
What about contact with other people?
Remember, frequent careful hand washing by you and by any persons with whom you have 
physical contact is the best way to prevent transmission of virus.  During the time you need to wear 
a bandage (for a minimum of 3 weeks after vaccination) there are several kinds of people with 

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whom you should avoid close contact.  "Close contact" means that you sleep in the same bed with 
the person, give the person baths, and/or touch their bare skin to change their clothes (or diapers), 
apply ointments, or change their bandages. 
The individuals you should avoid include children ≤ 3years of age; pregnant women or nursing 
women; individuals with eczema, history of eczema or other skin conditions such as active cases 
of extensive psoriasis, severe rashes, generalized itching, infections, burns, chicken pox, or skin 
trauma; and/or immune suppressed individuals such as individuals with leukemia or lymphoma, 
with AIDS, or those receiving immunosuppressive treatment (for example, after organ transplant).  
 5.
Who do I contact when I have a question?
If you have any questions at any time, please call.  A nurse or a physician is available 24 hours a 
day by telephone.  To speak with the research nurse, call the research nurse’s office during regular 
business hours; if your question is not urgent, you may leave a message for the research nurse on 
the answering machine.  If your question is urgent or if you have a question outside of the regular 
business hour, please contact the 3NW inpatient unit and speak with the charge nurse. 
In an emergency, call 911 or report to the local emergency room/urgent care center.  If you have to 
go to an emergency room, please ask the doctors to call NIH for more information.
PHONE NUMBERS
3NW Inpatient unit
(301) 451-0789
12th floor Oncology Clinic
(301) 496-4026
*after regular business hours, please contact 3NW inpatient unit.
 

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16.3
APPENDIX C: MANAGEMENT ALGORITHMS FOR ENDOCRINOPATHY, 
GASTROINTESTINAL, HEPATIC, NEUROLOGICAL, PULMONARY, RENAL, AND SKIN 
ADVERSE EVENTS 
16.3.1 Endocrinopathy Management Algorithm

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16.3.2 GI Adverse Event Management Algorithm

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16.3.3 Hepatic Adverse Event Management Algorithm

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16.3.4 Neurologic Adverse Event Management Algorithm

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16.3.5 Pulmonary Adverse Event Management Algorithm

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16.3.6 Renal Adverse Event Management Algorithm

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16.3.7 Skin Adverse Event Management Algorithm

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16.4
APPENDIX D: IMMUNE RELATED RESPONSE CRITERIA
Because of direct clinical observations of immune cell influx into tumor causing enlargement in 
some patients prior to sustained response, recently it has been suggested that clinical trials 
involving the use of immunotherapy use alternative guidelines, called immune related response 
criteria (irRC) to determine radiographic response or progression after therapy[49].  These 
recommendations have been used in recent clinical trials.  One study of 227 subjects with 
metastatic melanoma showed that the approximately 10% of patients who had PD by modified 
WHO criteria but either CR, PR or SD by irRC had a similar overall survival as those patients 
who had SD, PR or CR by both criteria. The irRC was created using bidimensional 
measurements (as previously widely used in the WHO criteria).  We have taken the concepts of 
the irRC and combined them with the recently revised RECIST 1.1[44] to come up with the 
modified irRC used in this protocol.  Consistent with the irRC, the main changes from RECIST 
1.1 are (a) a requirement for confirmation of both progression and response by imaging at least 4 
weeks after initial imaging and (b) not automatically calling the appearance of new lesions 
progressive disease if the total measurable tumor burden has not met criteria for progressive 
disease.  
For immune-related response criteria (irRC), only index and measurable new lesions are taken 
into account.  At baseline tumor assessment on this trial, target lesions will be measured along 
the longest axis and the measurements will be summed, called sum of largest diameter (SLD).  
These lesions must be a minimum of 10mm per lesion, maximum of 5 target lesions, maximum 
of 2 per organ system.  At each subsequent tumor assessment, the unidimensional measurement 
of target lesions and of new measureable lesions are added together to provide the total tumor 
burden:  As per the modified definitions below, all responses and progression except stable 
disease (SD) require confirmation on a consecutive scan at least 4 weeks from the initial 
observation). 
Definitions of irRC:
Response
irRC
New measurable lesions 
Incorporated into tumor burden
New non-measurable 
lesions 
Do not define progression (but precludes irCR)
Non-index lesions
Contributes to defining irCR (complete disappearance required)
Overall irCR
100% disappearance of all lesions, whether measurable or not, and no new lesions, in 
two consecutive observations not less than 4 wks from the date first documented.  All 
measurable lymph nodes also must have reduction in short axis to <10mm.
Overall irPR
≥ 30% decrease in SLD compared with baseline confirmed by a consecutive 
assessment at least 4 wk after first documentation
Overall irSD
Not meeting criteria for irCR or irPR, in absence of irPD:  30% decrease in SLD 
compared with baseline cannot be established nor 20% increase compared with nadir.
Overall irPD
At least 20% increase in SLD compared with nadir (minimum recorded tumor 

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burden) and an increase of at least 5mm over the nadir, confirmed by a repeat, 
consecutive observations at least 4 wk from the date first documented.
Overall responses derived from changes in index, non-index and new lesions as demonstrated in 
the following table:
Measurable response
Non-measurable response
Overall response 
using irRC
Index and new, measurable 
lesions (tumor burden)* %
Non-index lesions
New, non-measurable 
lesions
Decrease 100
Absent
Absent
irCR&
Decrease 100
Stable
Any
irPR&
Decrease 100
Unequivocal 
progression
Any
irPR&
Decrease ≥ 30%
Absent / Stable
Any
irPR&
Decrease ≥ 30%
Unequivocal 
progression
Any
irPR&
Decrease < 30 to increase < 20
Absent / Stable
Any
irSD
Decrease < 30 to increase < 20
Unequivocal 
progression
Any
irSD
Increase ≥ 20
Any
Any
irPD
* Decreases assessed relative to baseline  
&  Assuming response (irCR) and progression (irPD) are confirmed by a second, consecutive assessment at least 
4 weeks apart.