Phase II Trial of Combination Immunotherapy in Biochemically Recurrent Prostate Cancer
Phase: PHASE2
Conditions: Prostate Cancer
Sponsor: National Cancer Institute (NCI)



Abbreviated Title: Combination Immunotherapy 
Version Date:  05/10/2023
2
PRÉCIS
Background:

Androgen deprivation therapy (ADT) and surveillance are treatment options for prostate 
cancer patients with biochemical progression after localized therapy (i.e., biochemically 
recurrent [BCR] prostate cancer). The primary goal in these patients is to prevent 
morbidity from their cancer that results from disease progression and metastatic disease 
on conventional imaging. 

ADT can lower the PSA in these patients, but because of its substantial side effect profile 
and ambiguous long-term impact, it is generally deferred by most patients until there is a 
rapid escalation in their PSA. 

Immunotherapy presents an alternative option for these patients that is especially 
attractive because it is not associated with substantial toxicity. Also, since 
immunotherapy can have lasting effects after treatment due to a sustained activated 
immune response, patients will not be required to take these treatments indefinitely to 
potentially benefit clinically.

Current and previous clinical trials have demonstrated that single agent immunotherapy 
can impact PSA in patients in this population.

The focus of this study is to determine if combination immunotherapy with immune-cell 
mobilizing vaccines can initiate an immune response in the first 4 months that is then 
augmented by an immune checkpoint inhibitor in the following 3 months.

In addition to PSA responses (the primary metric in this population), safety, changes in 
immune responses, and PSA kinetics will also be evaluated. 
Objectives:
Primary Objectives:

Safety Lead-In: To evaluate the safety and tolerability of combination immunotherapy in 
participants with castration-resistant prostate cancer

Biochemical Recurrence: To determine if the combination immunotherapy can induce a 
30% decline in PSA in 28% of participants with biochemically recurrent prostate cancer. 
Eligibility Criteria (for biochemical recurrence):

Histologically confirmed adenocarcinoma of the prostate  

Participants with negative CT Scan and Tc-99m Bone Scan  

Participants with a PSA over 0.8 ng/ml for participants following radical prostatectomy 
or for participants following definitive radiation therapy: a rise in PSA of ≥ 2 ng/mL 
above the nadir 

Participants with a PSA doubling time of 5-15 months

No history of active autoimmune disease or history of organ compromising autoimmune 
disease

ECOG 0-1

Safety lead-in cohort will evaluate 6 participants with castration resistant prostate cancer

Abbreviated Title: Combination Immunotherapy 
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Design:

Three-arm, non-randomized study

Accrual goal is a total of 37 evaluable participants (6 in an initial safety cohort, 6 
participants who received M7824 as part of the initial investigation and 25 homogenously 
treated participants) to evaluate response

Participants from an on-going study (NCT02649439) with nearly identical eligibility can 
serve as a contemporary control for secondary endpoints

Following the safety lead-in, all participants will be enrolled and may undergo a 
surveillance period during which up to 4 consecutive monthly PSA values will be 
captured by the labs. 

After surveillance period, if applicable, participants will be treated with 2 vaccines 
concurrently, Prostvac and CV301, during months 1-4. For months 5-7, MSB0011359C 
[an anti-PD-L1 antibody (avelumab) with TGFβ-Trap molecule] will be added to the 
regimen.
o
Effective with amendment v7/29/2021, MSB0011359C will no longer be given as 
part of the treatment combination for the Biochemical Recurrence cohort (Arms 2 
and 3)

Participants will be monitored for on-treatment and post-treatment PSA, immune and 
imaging responses.

Abbreviated Title: Combination Immunotherapy 
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SCHEMA
Prostvac: Q2 weeks for 1 month, then monthly for 6 additional months (7 months total)
CV301: Q2 weeks for 1 month, then monthly for 6 additional months (7 months total)
MSB0011359C: Given q 2 weeks for 3 months at phase II dose (flat dose of 1200 mg) starting 
after 4 months of vaccines. Effective with amendment v7/29/2021, MSB0011359C will no 
longer be given as part of the treatment combination for the Biochemical Recurrence cohort 
(Arms 2 and 3). This change is reflected in the new cohort 2 schema above. 
o
With Amendment v05/10/2023, participants that have at least 3 PSA measurements in the 
surveillance period, with at least one of them sufficient to provide the doubling time from 
baseline, will not undergo any further PSA measurements in the surveillance period.
Phase 1 Lead-in:  6 participants treated with Prostvac+CV-301 and anti-PDL1/TRAP*

*Phase 1 dosing will be given concurrently in castration-resistant prostate cancer 
participants until disease progression or toxicity limits dosing

Participants in the Phase 1 Lead-in will not undergo the surveillance period

Abbreviated Title: Combination Immunotherapy 
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TABLE OF CONTENTS
PRÉCIS............................................................................................................................................2
SCHEMA.........................................................................................................................................4
TABLE OF CONTENTS.................................................................................................................5
STATEMENT OF COMPLIANCE.................................................................................................8
1
INTRODUCTION....................................................................................................................8
1.1
Study Objectives ...............................................................................................................8
1.2
Background and Rationale ................................................................................................8
2
ELIGIBILITY ASSESSMENT AND ENROLLMENT ........................................................24
2.1
Eligibility Criteria ...........................................................................................................24
2.2
Screening/Eligibility Evaluation .....................................................................................28
2.3
Participant Registration and Status Update Procedures ..................................................29
2.4
Baseline Evaluation.........................................................................................................29
3
STUDY IMPLEMENTATION ..............................................................................................30
3.1
Study Design ...................................................................................................................30
3.2
Drug Administration .......................................................................................................31
3.3
Treatment Modifications.................................................................................................33
3.4
Protocol Evaluation.........................................................................................................34
3.5
Cost and Compensation...................................................................................................36
3.6
Criteria for Removal from Protocol Therapy and Off Study Criteria.............................36
3.7
Follow-up Evaluations ....................................................................................................37
4
CONCOMITANT MEDICATIONS ......................................................................................37
4.1
Excluded Medications.....................................................................................................37
4.2
SUPPORTIVE CARE.....................................................................................................38
4.3
Treatment of vaccinia vaccination complication ............................................................38
5
BIOSPECIMEN COLLECTION............................................................................................39
5.1
Correlative Studies for Research.....................................................................................39
5.2
Collection of Specimens..................................................................................................42
5.3
Sample Storage, tracking, and Disposition .....................................................................43
5.4
Samples for Genetic/Genomic Analysis .........................................................................45
6
DATA COLLECTION AND EVALUATION ......................................................................46
6.1
Data Collection................................................................................................................46
6.2
Data Sharing Plans ..........................................................................................................47
6.3
Response Criteria ............................................................................................................48
6.4
Toxicity Criteria..............................................................................................................48

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7
NIH Reporting/Data and Safety monitoriing Plan..................................................................48
7.1
Definitions.......................................................................................................................48
7.2
OHSRP Office of Compliance and Training/IRB Reporting..........................................49
7.3
NCI Clinical Director Reporting.....................................................................................49
7.4
Institutional Biosafety Committee (IBC) Reporting Criteria..........................................49
7.5
NIH Required Data and Safety Monitoring Plan ............................................................50
8
SPONSOR Protocol/SAFETY REPORTING........................................................................51
8.1
Definitions.......................................................................................................................51
8.2
Assessment of Safety Events...........................................................................................52
8.3
Reporting of Serious Adverse Events .............................................................................53
8.4
Waiver Of Expedited Reporting To CCR .......................................................................53
8.5
Safety Reporting Criteria to the Pharmaceutical Collaborators......................................53
8.6
Reporting Pregnancy.......................................................................................................54
8.7
Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND...................54
8.8
Sponsor Protocol Deviation Reporting ...........................................................................54
9
CLINICAL MONITORING...................................................................................................55
10 STATISTICAL CONSIDERATIONs....................................................................................56
10.1
Study Objectives .............................................................................................................56
10.2
Sample size determination ..............................................................................................56
10.3
Populations for analysis ..................................................................................................57
10.4
Statistical Analyses .........................................................................................................57
11 COLLABORATIVE AGREEMENTS...................................................................................59
11.1
Collaborative Research and Development Agreement (CRADA)..................................59
12 HUMAN SUBJECT PROTECTIONS ...................................................................................59
12.1
Rationale for Subject Selection.......................................................................................59
12.2
Participation of Children.................................................................................................59
12.3
Participation of Subjects Unable to Give Consent..........................................................60
12.4
Evaluation of Benefits/Risks/Discomforts......................................................................60
12.5
Risks/Benefits Analysis...................................................................................................61
12.6
Consent Process and Documentation..............................................................................62
13 REGULATORY AND OPERATIONAL CONSIDERATIONS...........................................63
13.1
Study Discontinuation and Closure.................................................................................63
13.2
Quality Assurance and Quality Control ..........................................................................64
13.3
Conflict of Interest Policy ...............................................................................................64
13.4
Confidentiality and Privacy.............................................................................................64
14 PHARMACEUTICAL INFORMATION ..............................................................................65

Abbreviated Title: Combination Immunotherapy 
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14.1
Recombinant Fowlpox-PSA(L155)/TRICOM............................................................65
14.2
Recombinant Vaccinia-PSA(L155)/TRICOM............................................................68
14.3
CV301 .............................................................................................................................79
14.4
MSB0011359C (M7824, Bintrafusp Alfa)......................................................................81
15 REFERENCES .......................................................................................................................88
16 APPENDIX A: Performance Status Criteria..........................................................................91
17 APPENDIX B: Vaccinia-PSA(L155)/Tricom Patient Instruction Sheet................................92
18 APPENDIX C: On Study and Follow-Up Evaluations ..........................................................95
18.1
Study Calendar: Biochemical Recurrence Cohort ..........................................................95
18.2
Study Calendar: CRPC Lead-in Cohort ..........................................................................99
19 APPENDIX D: Instructions for Pre-Study and Follow-Up Blood Tests .............................102
20 APPENDIX F: Brown Top Specimen Collection Instructions.............................................103
21 APPENDIX G:  Purple Top Stool Collection Instructions...................................................105
22 APPENDIX H: Stool Specimen Shipment Instructions.......................................................106

Abbreviated Title: Combination Immunotherapy 
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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 Objectives

Safety Lead-In: To evaluate the safety and tolerability of combination immunotherapy in 
participants with castration-resistant prostate cancer

Biochemical Recurrence: Determine if the combination immunotherapy can induce a 30% 
decline in PSA in 28% of participants with biochemically recurrent prostate cancer. 
1.1.2
Secondary Objectives

Assess the safety of the combination immunotherapy regimen

Changes in PSA kinetics after treatment with the combination immunotherapy, relative to 
baseline PSA kinetics
1.1.3
Exploratory Objective

Immune changes after treatment with 2 vaccines for 4 months

Correlate changes in PET images to changes in PSA over time

Evaluate changes in the microbiome of the gastrointestinal tract and correlate to changes 
in treatment, PSA and immune cell subpopulations

Evaluate the decline in PSA in participants who received vaccine as well as MSB0011359B 
prior to its being discontinued.
1.2
BACKGROUND AND RATIONALE
1.2.1
Rationale for conducting the study in biochemically recurrent prostate cancer patients
Although surgery or radiation can cure the majority of newly diagnosed patients, 20-40% are likely 
to have recurrent disease manifested as a rising PSA despite negative conventional imaging studies 
(CT and technetium-99m (Tc-99m) Bone Scan) used to detect metastasis.[1, 2] This stage of 
disease is often referred to as biochemically recurrent prostate cancer.

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Figure 1:Poxviral vaccine strategy: Modified poxvirus contains transgenes for the tumor-associated antigen PSA and 3 T-cell 
costimulatory molecules [8, 9].
Figure 2:Prostvac improved survival in mCRPC patients in a randomized multi-center phase II trial. [11]
Prostvac is very well-tolerated, with common side effects of grade 1 injection-site reactions or flu-
like symptoms.[11, 12] A favorable side effect profile and the potential ability to induce a sustained 
antitumor immune response, clinical trials are ongoing evaluating strategies to use Prostvac in 
earlier disease patients. A previous study of Prostvac in biochemically recurrent prostate cancer 
suggested that this treatment could slow/improve PSA doubling in 25 patients from a median of 
5.3 months to 7.7 months.[13] A randomized prospective study is currently evaluating this 
hypothesis with the same treatment dosing and schedule as the previous study (NCT02649439). 
The treatment dosing and schedule for this combination immunotherapy study is the same as the 
previous studies.
1.2.4
CV301
Both CEA and MUC-1 have been reported as relevant immunologic targets in prostate cancer. 
CEA has been reported as elevated in the circulation of nearly 50% of 141 patients evaluated with 
advanced disease.[14] A separate study suggested that more than 60 percent of primary tumors 
have been found to express MUC1 and over 90% of metastatic lesions in lymph nodes have also 
been found to express MUC1.[15] Furthermore, more aggressive disease (higher Gleason Score) 
has also been associated with greater MUC1 expression.[15] Immunologic studies here at the NCI 
have demonstrated that despite initial targeting of PSA by pox virus-based vaccines, increased 
immunologic targeting of MUC1 and CEA have occurred.[16] Across several other trials, broad 
immunologic targeting of multiple antigens after immunotherapy has been associated with better 
clinical outcomes.[17]

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Earlier efforts to generate efficient cancer immunotherapy were directed towards the development 
of PANVAC, a prime and boost approach based on the administration of a replicating-competent 
Vaccinia-based delivery vector followed by a Fowlpox vector. Both vectors encoded the 
transgenes for B7.1, ICAM-1 and LFA-3, together with the CEA and MUC-1. In order to establish 
its therapeutic potential, several preclinical studies were performed, showing that PANVAC 
activates antigen-specific human T cells in vitro [18],elicits antibody and cellular immune 
responses in vivo, and shows significant antitumor efficacy against tumors expressing either human 
CEA or MUC-1 in mice, resulting in programmed death ligand 1 (PD-L1) up-regulation within 
the tumor microenvironment. Subsequently, the clinical development program continued with the 
design of several clinical trials to further confirm the efficacy and safety of this intervention. 
Overall, the results of Phase I and II clinical trials in different cancer populations employing 
PANVAC monotherapy or in combination with GM-CSF or docetaxel demonstrate a good safety 
profile accompanied by some hints of clinical benefits.[19, 20] 
A second-generation cancer immunotherapy strategy was developed, giving rise to CV301 
involving a modified vaccinia. CV301 is a recombinant non-replicating poxvirus-based 
immunotherapy encoding the same 5 transgenes used in PANVAC (B7.1, ICAM-1, LFA-3, CEA 
and MUC-1) and thus designed to activate an antigen-specific response. Therefore, tumors 
expressing these antigens, including prostate cancer, qualify as a potential indication for CV301. 
Given the potential risks associated with the replicating capacity of the vaccinia-based PANVAC-
V construct, the next generation cancer immunotherapeutic candidate MVA-BN-CV301 was 
generated 
 It constitutes a 
recombinant non-replicating poxvirus- based immunotherapy derived from the MVA-BN 
construct. This novel viral vector was developed from the highly attenuated vaccinia virus known 
as MVA, which after more than 500 serial passages in chicken embryo fibroblasts lost 
approximately 15% of the original vaccinia genome. Furthermore, MVA-BN differs from other 
MVA strains in its extensive plaque purification and propagation in serum-free conditions, and has 
already undergone extensive investigation as a new smallpox vaccine. Within this comprehensive 
clinical development program, MVA-BN has been administered to more than 7,700 subjects 
without major safety concerns, hence exhibiting a robust safety profile. Accordingly, besides 
decreasing the risk of cardiac adverse events associated with earlier generation vaccinia-based 
strategies, the improved MVA-BN enables previously excluded segments of the general 
population, such as immunocompromised individuals and persons diagnosed with atopic 
dermatitis, to receive the potential benefits offered by this novel approach. Therefore, it embodies 
a highly novel approach and offers a reliable technological platform that enables the safe delivery 
of different transgenes to produce a tailored immune response against specific antigens.
The new CV301 strategy encodes the same 5 transgenes expressed by the previous PANVAC 
vector: CEA, MUC-1, B7.1, ICAM-1 and LFA-3. However, these inserts encode each of the 
transgenes with amino acid sequences that are slightly different from the original vectors. In fact, 
MUC-1 has been modified with additional agonistic epitopes. Consequently, the CV301 construct 
is expected to promote an antigen-specific targeted immune response for tumors expressing these 
antigens. In addition, a change in biosafety also characterizes the new CV301 construct. Thus, 
whereas vaccinia possesses a BSL-2, MVA has been categorized as BSL1 in Europe. As well, 
similarly to the earlier PANVAC concept, CV301 also encompasses a heterologous prime-boost 
regimen employing an MVA-BN-CV301 priming dose followed by a Fowlpox-based FPV-CV301 
boosting dose, thus replacing the former PANVAC-V and PANVAC-F components. Given the 

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existing correspondences between the CV301 and PANVAC vectors, the clinical development 
program designed for CV301 will rely on the extensive nonclinical and clinical data currently 
available from PANVAC.
CV-301 Phase I Experience
A phase I study was just completed at the NCI with no dose limiting toxicities. (unpublished)  As 
expected, CV-301 was well tolerated in all 12 patients with no DLT noted. The only attributable 
toxicities were grade 1 and 2 and mostly injections site reactions and flu-like symptoms, with other 
rare toxicities such as headache and nausea (also grade 2 or less) also reported. There were no 
grade 3 or greater toxicities reported in the study. 
1.2.5
PD-L1 TGFβ Trap (MSB0011359C)
MSB0011359C is a bifunctional fusion protein that combines an anti-programmed death ligand 1
(PD-L1) antibody and the soluble extracellular domain of transforming growth factor beta (TGFβ) 
receptor type II as a TGFβ neutralizing “trap,” into a single molecule. This anti-PD-L1 / TGFβ-
Trap molecule is designed to target 2 major mechanisms of immunosuppression in the tumor 
microenvironment. The anti-PD-L1 moiety of MSB0011359C is identical to avelumab, except for 
three amino acid substitutions in the heavy chain constant regions, which result in a different 
human IgG1 allotype, and one amino acid substitution in the heavy chain for antibody stability. 
Avelumab (proposed international non-proprietary name for MSB0010718C) is currently in Phase 
II / III clinical development 
.
The PD-1/PD-L1 axis is an important mechanism for tumor immune evasion.[21] Effector T cells 
chronically sensing antigen take on an exhausted phenotype marked by PD-1 expression, a state 
under which tumor cells engage by upregulating PD-L1. Blocking the axis restores the effector 
function in these T cells. Additionally, in the tumor microenvironment, myeloid cells, 
macrophages, parenchymal cells, and T cells upregulate PD-L1. TGFβ has growth inhibitory 
effects on normal epithelial cells, functioning as a regulator of epithelial cell homeostasis, and it 
acts as a tumor suppressor during early carcinogenesis. As tumors progress toward malignancy, 
the growth inhibitory effects of TGFβ on the tumor are lost via mutation in one or more of the 
TGFβ pathway signaling components or through oncogenic reprogramming.[22] Upon loss of 
sensitivity to TGFβ inhibition, the tumor continues to produce high levels of TGFβ, which then 
serve to promote tumor growth.[22] The TGFβ cytokine is overexpressed in various cancer types 
with correlation to tumor stage.[22, 23] Many types of cells in the tumor microenvironment 
produce TGFβ, including the tumor cells themselves, immature myeloid cells, regulatory T cells, 
and stromal fibroblasts; these cells collectively generate a large reservoir of TGFβ in the 
extracellular matrix. TGFβ signaling contributes to tumor progression by promoting metastasis, 
stimulating angiogenesis, and suppressing innate and adaptive antitumor immunity.[22] As a 
broadly immunosuppressive factor, TGFβ directly down regulates the effector function of 
activated cytotoxic T cells and natural killer (NK) cells and potently induces the differentiation of 
naïve CD4+ T cells to the immunosuppressive regulatory T cells (Treg) phenotype.[23] In 
addition, TGFβ polarizes macrophages and neutrophils to a wound-healing phenotype that is 
associated with production of immunosuppressive cytokines.[24] As a therapeutic strategy, 
neutralization of TGFβ activity has the potential to control tumor growth by restoring effective 
antitumor immunity, blocking metastasis, and inhibiting angiogenesis. In prostate cancer, TGFβ 
has been associated in poor outcomes or disease recurrence in early stage patients, perhaps 
suggesting that it may be a therapeutic target ([25],[26]).

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MSB0011359C also binds TGFβ (preferentially 1 and 3 isoforms), an inhibitory cytokine 
produced in the tumor microenvironment by cells including apoptotic neutrophils, myeloid-
derived suppressor cells, T cells, and tumor.[21, 27] Inhibition of TGFβ by soluble TGFβRII 
reduced malignant mesothelioma tumors in a manner that was associated with an increase in CD8+ 
T cell antitumor effects.[28] The absence of TGFβ1 produced by activated CD4+ T cells and Treg 
cells has been shown to inhibit tumor growth, and protect mice from spontaneous cancer.[29]  
Thus, TGFβ appears to be important for tumor immune evasion. 
Combining these pathways, PD-1 / PD-L1 and TGFβ, is attractive as an antitumor approach. A 
recent report found that blockade of TGFβ signaling in T cells or deletion of TGFβ1 from T cells 
in a mouse model led to diminished PD-1 expression in tumor-infiltrating CD8+ T cells.[29] 
Concomitant PD-1 and TGFβ blockade can restore pro-inflammatory cytokines.[30] In a murine 
model of hepatocellular carcinoma, TGFβ appeared to increase the expression of PD-L1 in 
dendritic cells, which in turn promoted T-cell apoptosis and increased percentage of CD25+, 
Foxp3+ T regulatory cells.[31] Higher levels of circulating myeloid-derived suppressor cells 
(MDSCs), a significant source of TGFβ, are associated with failure to respond to anti-PD-1 
therapy.[32]
NCT02517398 is a phase 1, open label, 3+3 dose-escalation study and this preliminary data was  
presented at AACR in April, 2017. Eligible patients received MSB0011359C at 1, 3, 10, or 20 
mg/kg Q2W until confirmed progressive disease, unacceptable toxicity, or trial withdrawal; 
treatment beyond progression is generally allowable. The primary objective is to determine the 
safety and maximum tolerated dose of MSB001359C; secondary objectives include 
pharmacokinetics (PK), immunogenicity, and best overall response per RECIST v1.1. As of Oct 
3, 2016, 16 heavily pretreated pts with ECOG performance status 0-1 have received 
MSB0011359C. Our PK data show a dose-linear increase in exposure; furthermore, at the 
examined dose levels, MSB0011359C saturates peripheral PD-L1 and sequesters any released 
plasma TGF-β1, -β2, and -β3 throughout the dosing period. Grade 3 drug-related treatment-
emergent adverse events (TEAEs) occurred in 3 patients (skin infection secondary to grade 2 
bullous pemphigoid [BP], lipase increased, anemia, and colitis); there were no grade 4-5 drug-
related TEAEs. BP and colitis responded well to steroids. Colitis and its secondary events of 
anemia and rectal hemorrhage (in a previously radiated area) were considered dose-limiting in 1 
patient. (See full table of AEs in table below). There was preliminary evidence of efficacy across 
all dose levels, including 1 ongoing confirmed complete response (cervical cancer), 1 durable 
partial response (pancreatic cancer), a 25% reduction in the sum of diameters of target lesions after 
only 2 doses of MSB001359C (cervical cancer), and 2 cases of prolonged stable disease 
(pancreatic cancer; carcinoid). Updated data will be presented. Preliminary data from this phase 1 
dose-escalation study suggest that MSB0011359C has a manageable safety profile in patients with 
heavily pretreated advanced solid tumors. Early evidence of clinical efficacy warrants further 
study. The recommended phase 2 dose is a flat dose of 1200 mg every 2 weeks. 
 
n = 16 cohort
n = 3 backfill data
 Treatment Related Adverse 
Events (Gulley JL et al., ASCO 
2017)
Any 
Grade
Grade 3
Any 
Grade
Grade 3

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n = 16 cohort
n = 3 backfill data
Patients with any event, n (%)
7 (43.8)
3 (18.8)
2 (66.7)
Anemia
1 (6.3)
1 (6.3)
Bullous pemphigoid 
1 (6.3)
 
Colitis
1 (6.3)
1 (6.3)
Dermatitis acneiform
1 (6.3)
 
Dyspnea exertional
1 (6.3)
 
Hyperthyroidism
1 (6.3)
 
1 (33.3)
Hypophosphatemia
1 (6.3)
 
Hypothyroidism
2 (12.5)
 
1 (33.3)
Infusion-related reaction
1 (6.3)
 
Keratoacanthoma
1 (6.3)
 
1 (33.3)
Lipase increase
1 (6.3)
1 (6.3)
Nausea
1 (6.3)
 
Peripheral motor neuropathy
1 (33.3)
Pruritus
1 (6.3)
 
Rash maculopapular
2 (12.5)
 
1 (33.3)
Skin infection
1 (6.3)
1 (6.3)
Vomiting
1 (6.3)
 
As of 24 August 2018, clinical activity of MSB0011395C has been observed in the dose 
escalation cohorts of EMR200647-001. For the 40 participants analyzed at the 1200mg level, 
Overall Response Rate (ORR) assessed by the Investigator was 25%, with median progression-
free survival (PFS) of 2.7 months and overall survival of 17.1 months (data cutoff 15 October 
2019). 
1.2.6
Rationale to Combine Vaccines
Previous studies have demonstrated that in vivo broadening of the immune response through 
targeting of additional antigens after immunotherapy can lead to better outcomes. This process is 
called antigen spreading or antigen cascade.[33] Although this has been seen and reported 
previously with single vaccine administration, it is possible that in vivo broadening of the immune 
response could be augmented by the use of multiple vaccines.

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Preclinical models support this hypothesis using two vaccines in a female C57BL/6 (H-2b) mouse 
model.[34] These studies demonstrated that concurrent administration of two vaccines induced a 
more diverse T-cell population that lead to enhanced antitumor efficacy. These studies provide the 
rationale for clinical studies investigating concurrent administration of vaccine platforms to 
enhance the antigen-specific immune response. This strategy has not been tested in prostate cancer 
to this point and this study will be the first to explore this strategy and its impact immunologically 
and on PSA in biochemical recurrence. Given that both vaccines are well tolerated with negligible 
side effect profiles, there are limited concerns about toxicity from this combination.
1.2.7
Rationale to Combine Immune Checkpoint Inhibitors with Vaccines
To this point, immune checkpoint inhibitors have failed to demonstrate clear single agent efficacy 
in prostate cancer. Two studies with ipilimumab failed to demonstrate an overall survival in phase 
3 studies.[35, 36] Anti-PD1 and anti-PDL1 have demonstrated only limited benefits. The study 
that has generated the most interest to this point involves pembrolizumab in mCRPC.[37] That 
study has reported a response rate of about 15-20% in the first 30 patients, typical of findings 
across multiple cancer subtypes. In order to enhance response to anti-PD1/anti-PDL1 inhibition, 
combinations with other therapies that can mobilize immune cells to the tumor microenvironment 
have been proposed.[38] In this manner, immune cells in the tumor microenvironment could be 
enabled by PD1/PDL1 inhibition.
This is likely the underlying mechanism for the approved ipilimumab /nivolumab combination in 
metastatic melanoma.[39] Anti-CTLA-4 (ipilimumab) is able to activate immune cells in the 
periphery and within tumor, leading to enhanced outcomes in combination with anti-PD1 
(nivolumab), improving progression free survival (PFS) in metastatic melanoma relative to 
nivolumab alone (11.5 vs. 6.9 months; p<.001) and ipilimumab alone (2.9 months, p<0.001). 
Interestingly a subgroup analysis from this study indicated that patients with PDL-1 positive 
tumors had no benefit from the combination vs. nivolumab alone (PFS of 12.4 vs. 12.4 
months).[39] This data would suggest that for patients lacking immune cells  in the tumor 
microenvironment (devoid of PD-L1 upregulation) benefited from ipilimumab induced immune 
mobilization from the periphery.[38] The toxicity of ipilimumab in prostate cancer patients limits 
its use in biochemical recurrence, but it is possible that vaccine combinations could accomplish 
the same results.
Indeed, preclinical models from the LTIB have suggested that vaccine-based therapies can lead to 
upregulation of PDL1 in the tumor microenvironment (unpublished). As demonstrated below, in 
murine model with CEA transgenic mice, a CEA vaccine led to upregulation of PDL1 expression. 
Mechanistically, the CEA vaccine likely increased immune cells in the tumor microenvironment 
after immune activation ultimately leading to increased PDL1 expression in the tumor.

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Preclinical investigations with a therapeutic cancer vaccine, TEGVAX, help mechanistically 
illustrate this hypothesis. TEGVAX has demonstrated the ability to increase interferon-gamma 
production in multiple murine models.[40] One series of experiments showed that PDL1 
upregulation on tumors was interferon-gamma dependent. Another set of experiments 
demonstrated synergistic anti-tumor activity when TEGVAX was combined with anti-PD1 
antibody. This benefit, however, was mitigated when an interferon-gamma blocking antibody was 
added to the model, demonstrating its integral role.
The sequence of the anti-PDL1 therapy in this study will allow for priming of the immune system 
and the perhaps “inflame” the tumor microenvironment prior to potentiation of a clinical response 
with the anti-PDL1 therapy. Although this treatment will only be given for 4 months, it should 
provide sufficient time for proof-of-concept for the efficacy of this therapy. 
1.2.8
Safety
Safety is a paramount concern in this population of patients who may live another decade. Vaccines 
have been frequently studied in this population of biochemical recurrence with minimal toxicities 
of injection site reactions and transient flu like symptoms. We do not expect combination vaccine 
therapy to induce more sustained toxicities. This is an extrapolation based on data where vaccines 
have been associated with vivo expansion, resulting in the targeting of multiple antigens (antigen 
spread/antigen cascade) and such patients often have better clinical outcomes and are not 
associated with greater toxicity.[16] [41] The addition of an immune checkpoint inhibitor does add 

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potential for greater toxicities but these toxicities should be manageable as demonstrated in other 
clinical trials using checkpoint inhibitors as monotherapy. 18 patients with mCRPC were treated 
with avelumab (the anti-PDL1 antibody that is the backbone of MSB0011359C) and the toxicities 
are reported in Table 1. Immune-mediate adverse events are the greatest concern for these patients, 
and patients will have detailed informed consent discussions on this manner. 
Reported Toxicities in mCRPC patients 
treated with Avelumab (n=18)
Maximum grade, (%)
1
2
3
4
All
13 (72%)
8 (44%)
2 (11%)
0
Fatigue
5 (28%)
0
0
0
Joint pain
1 (5%)
0
0
0
Infusion reaction
0
4 (22%)
0
0
Rash
2 (11%)
0
0
0
Hypothyroidism
1 (5%)
3 (17%)
0
0
Elevated liver 
enzymes
0
2 (11%)
0
0
Amylase/Lipase 
elevation
0
1 (5%)
2 (11%)
0
Table 1: Reported toxicities in mCRPC patients treated with avelumab (the anti-PDL1 antibody backbone of 
MSB0011359C). 
Nonetheless, out of an abundance of caution the three agents will be used together in a 6 patient 
phase I lead in cohort in advanced (castration resistant prostate cancer). Once all 6 patients have 
completed a 6-week toxicity assessment period the toxicity data will be shared with IRB before 
moving for with the planned study in biochemical recurrence.
Please note: As of December 21, 2018, the 6 patients in the lead in cohort have completed the 6-
week toxicity assessment period and no SAEs were seen. Per the protocol design patients will now 
be enrolled to the BCR cohort. The toxicity assessment of patients in the lead-in cohort is described 
in section 1.2.13.

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1.2.9
Evaluating the Immunologic Response
Our group in the LTIB has previously evaluated immunologic parameters in clinical trials with 
Prostvac among several immunotherapy studies.[12, 42] While these findings are not surrogate 
markers of response, they have improved our knowledge of a vaccine-generated immune response 
and provided a better understanding of what factors are potentially important in mounting a 
sufficient anti-tumor immune response that could be associated with improved clinical outcomes. 
These and other data may allow optimization of vaccines in subsequent clinical trials. Few groups 
are as well positioned and have the experience as the LTIB to conduct this rigorous immune testing 
from clinical samples of patients treated with Prostvac.
Antigen-Specific Immune Responses. While many clinical trials have been done in biochemically 
recurrent 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 patients 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. (Figure 3). This more extensive 
analysis will provide a more thorough understanding of the immune response initiated by Prostvac 
and a greater opportunity for clinical correlations. This type of analysis has not been previously 
prospectively performed in biochemically recurrent prostate cancer patients. 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 2).
The rationale for evaluating intracellular cytokine staining is that it is one method to demonstrate 
to what extent the immune system has been specifically activated by an immunotherapy (vaccine) 
to target specific tumor antigens. 
Figure 3: Overlapping PSA Peptide Assessment. This innovative approach to evaluating immune response allows for 
assessment of immune response to the entire PSA protein using overlapping 15 amino acid sequences. (Previous techniques have 
focused on assessing just one of these peptides).

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Table 2: 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. When only 
one peptide was used to analyze the immune responses, no immune responders were identified (unpublished).
Briefly, the methodology takes cryopreserved peripheral blood mononuclear cells (PBMCs) from 
patients before therapy and at the time of restaging. They are thawed and rested overnight at 37oC, 
5% CO2 in complete media (IMDM supplemented with 10% Human AB, 2mM glutamine, 100 
units/ml penicillin, and 100 μg/ml streptomycin). The next day (Day 0), PBMCs are seeded in 12 
well plates (2.5 x106 in 1 mL), and stimulated with peptide mixes (0.1ug/mL per peptide); cultures 
were supplemented on days 3 and 5 with cytokines (IL7 and IL15, 10 ng/mL, PeproTech, Rocky 
Hill, NJ) and fresh media, and on day 7 are rested (with removal of cytokine and peptide). On day 
11, 1x106 cells were restimulated for 24 hours in 96 well plates with peptide mixes in the presence 
of anti-CD107a-APC (clone H4A3, BD Biosciences); brefeldin A (1ul/mL) and monensin 
(0.7ul/mL) (BD Biosciences) are added to cultures 2 hours after the start of the restimulation and 
incubated for the final 22 hours. PBMCs are then stained with anti-CD4-PerCP-Cy5.5 (clone 
OKT4, Biolegend), anti-CD8-AF700 (clone OKT8, Ebioscience), and anti-TNF-PE (clone 
MAb11), anti-IFNγ-PE-Cy7 (clone 4SB3), and anti-IL-2-BV521 (clone 5344.111) (BD 
Biosciences). At least 3x105 events in the live gate are acquired with a BD LSR-II flow cytometer 
equipped with a UV, violet, blue, and red laser. FCS files are analyzed with FlowJo V.9.7 for 
Macintosh (TreeStar, Ashland, OR). Fluorescence minus one (FMO) controls are used for gaiting, 
and non-viable cells were excluded. The absolute number of CD4+ or CD8+ lymphocytes 

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Table 3: Analysis of Peripheral Immune Cells
Figure 5:  Preliminary Data of Immunoscore Associations with Clinical Outcomes. This data (unpublished) from a recently 
completed breast cancer study involving a therapeutic cancer vaccine highlights the potential of Immunoscore. In this study, 
patients with peripheral immunoscore (H, more than 13 parameters positive, measured prior to therapy) had a significant increase 
in progression free survival. This difference was only observed in the vaccine arm and not in the standard of care alone arm. A 
separate assessment is required in prostate cancer and will be done in this study. If similar findings are seen, the Immunoscore 
could be prospectively evaluated in future trials using the data from this study to establish which immune subsets are most 
relevant. 

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1.2.10 Rational for Determining PSA Response
It has been noted previously that immunotherapy may have delayed clinical impact and 
preliminary data with prostvac alone in this population supports that hypothesis. This data comes 
from the ongoing NCI study: Prostvac in Patients with Biochemically Recurrent Prostate Cancer 
(NCT02649439). Of the first 20 patients enrolled, 18 are evaluable for PSA responses >9 months 
after enrollment (2 patients withdrew from the study and are not included). In a preliminary 
analysis, those patients that ultimately experienced a PSA decline had an intra study apex PSA 
determined. The intra study apex PSA is defined as the maximum on-study PSA that can be 
corroborated by another on-study PSA value within 10%. PSA values beyond 10% may represent 
lab error and therefore are not used to determine the intra-study apex (see Figure 6 below).
Figure 6:
Of those 18 evaluable patients, 5 (27.8%) have had confirmed (at least 2 consecutive) PSA declines 
from an intra-study apex PSA with 3 declines being confirmed at ≥30% (see Figure 7). Two additional 
patients have unconfirmed declines, but are still being followed for confirmation. It is of note 
because of the 10 patients on the study thus far who had monthly PSAs evaluated as part of a 
protocol mandated surveillance cohort, no patients had any similar confirmed declines >10%. 
These findings suggest that late effects of the vaccine may impact PSA in this population. 
PSA declines similar to this have not been reported previously in this population with 
immunotherapy, but this approach (using the intra-study apex PSA) may be a reasonable way to 
assess clinical impact of immunotherapy in this setting. Correlative immune data could also 
support biomarker development in this population and have broader applications to other types of 
cancer. This proposed trial of combination immunotherapy in biochemically recurrent prostate 
cancer represents a reasonable follow-up to the current study (expected to complete accrual at the 
NCI by Quarter 3, 2017).

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Figure 7:
1.2.11 Rationale for Research PET Scans 
When logistically feasible, PET imaging will be done on patients in the BCR cohort as part of this 
study to evaluate for possible changes in PET images over time as they relate to PSA. In addition, 
such imaging could help identify patient who may have responses (e.g., patients with negative 
scans on other imaging, less tumor/less bone-based disease). 
1.2.12 18F-DCFPyL-PET/CT
PET imaging based on prostate specific membrane antigen (PSMA), including use of the 
radiotracer DCFPyL (Pylarify; manufacturer: Lantheus), which binds to PSMA, has emerged as 
a sensitive modality to detect localized and metastatic prostate cancer. 18F-DCFPyL is a second 
generation, high affinity agent that has shown outstanding efficacy in detecting prostate cancer, 
that received FDA approval in May 2021. 
18F-DCFPyL appears to be a sensitive and specific modality for staging prostate cancer and  
molecular analyses are underway as correlates to previous trials to identify which patients are 
most likely to be exceptional responders. Alternatively, early 18F-DCFPyL PET/CT scan (e.g., 
after ~2 months) may predict which patients go on to have exceptional responses. Thus, there is 
clinical benefit that could be derived from the ability of  18F-DCFPyL PET/CT imaging to 
monitor response to therapy.
1.2.13 Toxicity Assessment of Patients in Lead-In Cohort 
As planned, 6 patients were enrolled in the lead in cohort. As of December 21, 2018, the first 
three patients were removed from treatment for Progressive Disease. All six patients have 
completed the 6-week toxicity assessment period and no SAEs were seen. Additionally, none of 
the toxicities listed under the early stopping rules in section 8.4.1 have occurred on this trial. Of 
note, an asymptomatic increased Lipase - Grade 3, was captured on subject #1; treatment was not 
held as per protocol section 3.3.3. The AE resolved on its own without clinical symptoms. In 
addition, patient 6 had a brief episode of hematochezia (but no associated diarrhea). Out of an 
abundance of caution he was evaluated with a colonoscopy that demonstrated internal 
hemorrhoids and diverticulitis but no findings consistent with autoimmune colitis. The patient 
then continued all therapies. In addition, there have been no unexpected AEs to this point. At this 

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point, there is too little experience to make an estimation of clinical benefit, but there have been 
no unexpected safety signals therefore, we will initiate enrollment of BCR cohort per protocol 
design.
1.2.14 Rationale for removing MSB0011359C (M7824) 
We have decided to remove Bintrafusp Alfa from this study based on a global assessment of the 
risks and benefits of this agent in this population of cancer patients who have an expected 
median survival of approximately 10 years. Through the first six patients treated with this agent 
in the biochemical recurrence (main cohort), we saw one case of colitis and one case of 
myocarditis. The latter required extensive steroid dosing to resolve and the patient continues to 
have residual side effects from the high doses of steroids. Furthermore, through the first 6 
patients in biochemical recurrence cohort and the first 6 patients of castration resistant prostate 
cancer, there was no clear clinical impact of the Bintrafusp Alfa in any of the patients (no 
radiographic changes or PSA declines). The use of Bintrafusp Alfa was reviewed by the Clinical 
Immunotherapy Team here at the NCI in a specific toxicity meeting in May 2021.
Treatment regimen without an immune checkpoint inhibitor was considered to be optimal for 
patients of castration resistant prostate cancer. Given the longevity of this population, the 
favorable response without immune check inhibitor and the life expectancy of this population 
assessing a risk/benefit analysis, it was decided that Bintrafusp Alfa will be removed.
The rationale leading to this decision was discussed and approved 
.
2
ELIGIBILITY ASSESSMENT AND ENROLLMENT
2.1
ELIGIBILITY CRITERIA 
2.1.1
Inclusion Criteria for All Subjects
2.1.1.1
Histopathological documentation of prostate cancer confirmed in either the Laboratory 
of Pathology at the National Institutes of Health (NIH) Clinical Center, or Walter Reed 
National Military Medical Center prior to enrollment. If no pathologic specimen is 
available, participants may enroll with a pathologist’s report showing a histologic 
diagnosis of prostate cancer and a clinical course consistent with the disease. 
2.1.1.2
Recovery to baseline from acute toxicity related to prior therapy, including surgery and 
radiation (28 days removed from last systemic therapy, 14 days removed from last 
radiation therapy).

Hepatic function eligibility parameters (within 16 days before starting therapy):
o
Bilirubin ≤ ULN (OR in participants with Gilbert’s syndrome, a total bilirubin ≤ 
3.0), AST and ALT ≤ 1.5 times upper limit of normal.

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2.1.1.3
Adequate renal function defined by an estimated creatinine clearance > 50 mL/min 
according to the Cockcroft-Gault formula or by measure of creatinine clearance from 24 
hour urine collection.
2.1.1.4
No other active malignancies within the past 36 months (with the exception of 
nonmelanoma skin cancers or carcinoma in situ of the bladder) or life-threatening 
illnesses.
2.1.1.5
Willing to travel to the NIH for follow-up visits.
2.1.1.6
18 years of age or older. 
2.1.1.7
Able to understand and sign informed consent.
2.1.1.8
The effects Prostvac and CV301 on the developing human fetus are unknown. For this 
reason, men must agree to use highly effective contraception (that is, methods with a 
failure rate of less than 1% per year) prior to study entry, for the duration of study 
therapy and at least four months after the last treatment administration. Should a woman 
become pregnant or suspect she is pregnant while her partner is participating in this 
study, she should inform her treating physician immediately.
2.1.2
Additional Inclusion Criteria Specific to Safety 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: 
i. Radiographic progression defined as any new or enlarging bone lesions or growing 
lymph node disease, consistent with prostate cancer 
OR
ii. 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

ECOG performance status of 0–2 (Karnofsky >80%, see APPENDIX A).

Hematological eligibility parameters (within 16 days before starting therapy; see 
APPENDIX D):
o
Granulocyte count ≥ 1000/mm3 
o
Platelet count ≥ 100 000/mm3
o
Hgb ≥ 9 g/dL
o
PT ≤ 1.5 x ULN
o
aPTT ≤ 1.5 x ULN

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2.1.3
Additional Inclusion Criteria Specific to Biochemical Recurrence Cohort
2.1.3.1
Biochemical progression defined as follows:

For participants following definitive radiation therapy: a rise in PSA of ≥ 2 ng/mL 
above the nadir (per RTOG-ASTRO consensus criteria)

For participants following radical prostatectomy: rising PSA after surgical procedure 
(participants must have a PSA ≥ 0.8 ng/mL) 
2.1.3.2
Participants must have a rising PSA as confirmed by 3 values a minimum of 1 week 
apart over at least a 1 month period of time.
2.1.3.3
Participants must have a PSA doubling time of 5-15 months.
2.1.3.4
ECOG performance status of 0–1 (Karnofsky > 80%, see APPENDIX A).
2.1.3.5
Negative CT scan/MRI and bone scan for metastatic prostate cancer.
2.1.3.6
Baseline testosterone ≥ 100 ng/dl.
2.1.3.7
PSA ≤ 30 ng/mL.
2.1.3.8
Hematological eligibility parameters (within 16 days before starting therapy; see 
APPENDIX D):

Granulocyte count ≥ 1000/mm3 

Platelet count ≥ 100 000/mm3

Hgb ≥ 10 g/dL
2.1.4
Exclusion Criteria
2.1.4.1
Immunocompromised status due to:

Human immunodeficiency virus (HIV) positivity.

Active autoimmune diseases such as Addison's disease, Hashimoto's thyroiditis, 
systemic lupus erythematosus, Sjogren syndrome, scleroderma, myasthenia gravis, 
Goodpasture syndrome or active Grave's disease. 

Participants with a history of autoimmunity that has not required systemic 
immunosuppressive therapy or does not threaten vital organ function including 
CNS, heart, lungs, kidneys, skin, and GI tract will be allowed.

Participants with diabetes type I, vitiligo, or alopecia are allowed. 

Other immunodeficiency diseases

Splenectomy
2.1.4.2
Receipt of any organ transplantation, including allogeneic stem-cell transplantation, but 
with the exception of transplants that do not require immunosuppression (e.g., corneal 
transplant, hair transplant).
2.1.4.3
Chronic administration (defined as daily or every other day for continued use > 14 days) 
of systemic corticosteroids within 28 days before the first planned dose of 

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investigational therapy. Use of corticosteroids with minimal systemic absorption (e.g., 
inhaled steroids, nasal sprays, and topical agents) is allowed.
2.1.4.4
Serious intercurrent medical illness that, in the judgment of the investigator, would 
interfere with participant’s ability to carry out the treatment program.
2.1.4.5
Other medications used for urinary symptoms including 5-alpha reductase inhibitors 
(finasteride and dutasteride) and alternative medications known to alter PSA (e.g., 
phytoestrogens and saw palmetto).
2.1.4.6
History of prior chemotherapy (chemotherapy allowed for lead-in cohort in castration 
resistant disease.)
2.1.4.7
History of prior immunotherapy within the last 3 years (immunotherapy allowed for 
lead-in cohort in castration resistant disease.)
2.1.4.8
Receipt of an investigational agent within 28 days (or 56 days for an antibody-based 
therapy) before the first planned dose of study drugs.
2.1.4.9
Major surgery within 4 weeks prior to enrollment
2.1.4.10 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to poxviral vaccines (e.g., vaccinia vaccine) 
2.1.4.11 Previous serious adverse reactions to smallpox vaccination 
2.1.4.12 History of allergic reactions attributed to monoclonal antibodies (grade ≥ 3)
2.1.4.13 Known allergy to eggs, egg products, aminoglycoside antibiotics (e.g., gentamicin or 
tobramycin).
2.1.4.14 History of atopic dermatitis or active skin condition (acute, chronic, exfoliative) that 
disrupts the epidermis
2.1.4.15 Unable to avoid close contact or household contact with the following high-risk 
individuals for three weeks after the Day 1 vaccination: (a) children ≤ 3 years of age, (b) 
pregnant or nursing women, (c) individuals with prior or concurrent extensive eczema 
or other eczemoid skin disorders, or (d) immunocompromised individuals, such as those 
with HIV.
2.1.4.16 Participants who test positive for HBV or HCV  
2.1.4.17 Clinically significant cardiovascular/cerebrovascular disease as follows: cerebral 
vascular accident/stroke (< 6 months prior to the first planned dose of study drugs), 
myocardial infarction (< 6 months prior to the first planned dose of study drugs), 
unstable angina, congestive heart failure (New York Heart Association Classification 

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Class ≥ II), serious cardiac arrhythmia, or uncontrolled hypertension (SBP>170/ 
DBP>105).
2.1.4.18 Participants who have received a red cell transfusion within 2 weeks prior to 
enrollment.
2.1.4.19 Participants unwilling to accept blood products as medically indicated. 
2.1.4.20 Individual tumor lesion(s) in the liver or chest which are 10 cm or larger.
2.1.5
Recruitment Strategies
This protocol may be abstracted into a plain language announcement posted on NIH websites and 
on NIH social media platforms. This study will be listed on available websites 
(www.clinicaltrials.gov, 
https://www.cancer.gov/about-cancer/treatment/clinical-trials/search) 
and participants will be recruited from the current patient population at NIH.
2.2
SCREENING/ELIGIBILITY EVALUATION
Note: Screening evaluation testing/procedures are conducted under the separate screening 
protocol, 01-C-0129 (Eligibility Screening and Tissue Procurement for the NIH Intramural 
Research Program Clinical Protocols).
A. Pathological confirmation of diagnosis by either the Laboratory of Pathology at the NIH 
or Walter Reed National Military Medical Center may be obtained any time prior to 
enrollment (if specimen is available). If no pathologic specimen is available, participants 
may enroll with a pathologist’s report showing a histologic diagnosis of prostate cancer 
and a clinical course consistent with the disease.
B. The following parameters will be obtained within 8 weeks prior to start of enrollment:
1. HIV test (anti-HIV 1/2 antibody) 
2. Hepatitis B and C (HBs AG screening and anti-HCV antibody)
3. Tc-99 whole-body scintigraphy 
4. CT (or MRI may be substituted at investigator’s discretion) of chest, abdomen and pelvis
C. The following parameters will be obtained within 16 days prior to start of enrollment:
1. History and physical examination with ECOG 
2. Serum PSA 
3. Complete blood count plus differential, and platelet count 
4. Hepatic Panel (Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct 
Bilirubin)
5. Acute Care Panel (Sodium [NA], Potassium [K], Chloride [CL], Total CO2 
[Bicarbonate], Creatinine, Glucose, Urea Nitrogen, eGFR) 
6. Testosterone level
7. 24-hour urine collection if creatinine clearance to be confirmed by this method

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2.3
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.
2.3.1
Treatment Assignment Procedures (for registration purposes only)
Cohorts
Number
Name
Description
1
Lead-In Cohort  (closed 
December 2018)
Participants with advanced castration resistant prostate cancer 
(first six participants)
2
Biochemical Recurrence 
Cohort
Participants with biochemically recurrent prostate cancer
Arms
Number
Name
Description
1
Combination therapy  (closed 
December 2018)
Prostvac + CV301+ MSB0011359C
2
Combination therapy + 
surveillance (closed)
Surveillance followed by Prostvac + CV301 then Prostvac + 
CV301 + MSB0011359C
3
Combination vaccine 
therapy+/- surveillance
Surveillance as needed followed by Prostvac + CV301
Arm Assignment 
Participants in cohort 1 will be directly assigned to Arm 1 of the study. This cohort has been closed 
as of December 2018.
Participants in cohort 2 will be directly assigned to Arm 2, or Arm 3  of the study based upon time 
of enrollment. Participants enrolling after approval of amendment dated 7/29/2021 are assigned to 
Arm 3. 
Please note: As of December 21, 2018, the 6 participants in the lead in cohort have completed the 
6-week toxicity assessment period and no SAEs were seen. Per the protocol design participants 
will now be enrolled to the BCR cohort. The toxicity assessment of participants in the lead-in 
cohort is described in section 1.2.13.
2.4
BASELINE EVALUATION
A. The following parameters will be obtained within 8 weeks prior to enrollment:
1. Baseline electrocardiogram (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. 
B. The following parameters will be obtained within 16 days prior to start of treatment (these 
tests will not need to be repeated if they were done at screening within the appropriate 
timeframe):
1. Clinical evaluation

History and physical examination 

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
ECOG performance status (see APPENDIX A)

Height, weight
2. Laboratory studies

Serum PSA 

Serum testosterone level

CBC/differential, with platelet count

Serum chemistries (Na+, K+, Cl-, CO2, glucose, BUN, creatinine, albumin,
calcium, magnesium, phosphorus, alkaline phosphatase, ALT, AST, total 
bilirubin, LDH, CK, uric acid, total protein)

Serum amylase and lipase

Serum thyroid stimulating hormone (TSH)

Lymphocyte phenotyping CD3/CD4/CD8

Urinalysis in participants unless not feasible (i.e.. participant has incontinence)

Prostate acid phosphatase (PAP)
3
STUDY IMPLEMENTATION
3.1
STUDY DESIGN 
This is a three-arm, open-label trial of combination immunotherapy in participants with 
biochemical recurrent prostate cancer. 37 evaluable participants will be enrolled (after the Safety 
Lead-In cohort with 6 participants (arm 1), 6 participants who received M7824 as part of the 
initial investigation for PSA response (arm 2), and 25 homogenously treated participants (arm 3) 
with an accrual ceiling of 40 participants. 
A 6-participant phase I lead in cohort in advanced castration resistant prostate cancer participants 
will be conducted to determine safety. Dose levels for the lead in cohort are equivalent to the 
biochemical recurrence cohort. Participants will not have an observation period for the safety 
lead-in; dosing will begin after eligibility is confirmed (see Study Calendar in Section 18.2). 
Once all 6 participants have completed a 6-week toxicity assessment period the toxicity data will 
be shared with IRB before moving forward with the planned study in biochemical recurrence. 
Participants may remain on treatment with vaccines in the lead in cohort as long as the 
participant is willing and their disease remains stable on follow-up imaging. As of December 21, 
2018, the 6 participants in the lead in cohort have completed the 6-week toxicity assessment 
period and no SAEs were seen; therefore this cohort is closed.
Participants enrolled in Cohort 2 will undergo surveillance with four consecutive monthly PSA 
checks followed by Prostvac and CV301 during months 1-4 then Prostvac and CV301 during 
months 5-7. 
o
Existing participants with biochemical recurrent prostate cancer undergoing surveillance 
and PSA monitoring at the NIH may be enrolled at any point during the surveillance period 
provided the PSA monitoring is consistent with PSA monitoring in the Study Calendar 
(APPENDIX C) and the participant meets all other eligibility criteria. For example, if an 
eligible participant has 3 monthly PSA values, he may be enrolled onto the study, obtain 
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begin C1D1 with vaccine therapy. In this case, the 3 PSA values obtained prior to 
enrollment will be evaluated as if they were collected during the surveillance period while 
enrolled and will factor into the determination of the intra-study apex value (See Section 
6.3.2) highest PSA that is within 10% of another peak PSA for the participant on-study. 
Any other peak PSA values that are beyond 10% of other PSA values on the study will be 
determined as potential lab variability and excluded from response assessment.
o
With Amendment v05/10/2023, participants that have at least 3 PSA measurements in the 
surveillance period, with at least one of them sufficient to provide the doubling time from 
baseline, will not undergo any further PSA measurements in the surveillance period. 
Prostvac will be administered nearly identical to the Phase III dosing with Prostvac-V given day 
1 (week 1) followed by Prostvac-F on weeks 3 and 5 then monthly for an additional 5 months. 
Therefore, Prostvac-V is given one time (priming vaccine) followed by 7 doses of Prostvac-F 
(booster vaccine). The administration schema for CV301 is representative of the Phase I dose 
escalation trial. MVA-BN-CV301 (priming vaccine) is administered on day 1 (week 1) and week 
3 followed by FPV-CV301 (booster vaccine) administered on week 5 then monthly for an 
additional 5 months. 
MSB0011359C (anti-PDL1 TGFβ Trap) will be administered at the recommended phase II dose 
of 1200mg by IV infusion every 2 weeks for 3 months (6 doses) starting after 4 months of 
concurrent Prostvac and CV301.
Effective with amendment v7/29/2021, MSB0011359C will no longer be given as part of the 
treatment combination for the Biochemical Recurrence cohort (Arms 2 and 3). This cohort will 
now receive Prostvac and CV301 during months 1-7 following surveillance. 
3.1.1
Safety Assessment
For safety purposes, a minimum of 6 weeks must elapse between C1D1 of the third participant 
enrolled and C1D1 of the fourth participant enrolled which will allow for an adequate toxicity 
evaluation period of the combination therapies (i.e., 3 participants will have received combination 
vaccine and anti-PDL1 TGFβ Trap before any additional participants will be treated with that 
combination).
3.2
DRUG ADMINISTRATION
3.2.1
Prostvac

The Prostvac vaccine regimen consists of Prostvac-V (priming vaccine) and Prostvac-F 
(booster vaccine).

Prostvac will be administered nearly identical to the Phase III regimen with Prostvac-V 
(2x108 infectious units per 0.5 mL) given day 1 (week 1) followed by Prostvac-F (1x109 
infectious units per 0.5 mL) on weeks 3 and 5 then monthly for an additional 5 months. 

Prostvac is administered subcutaneously preferably in the upper thigh. Alternate 
subcutaneous sites may be used with approval of the principal investigator.

Prostvac 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. 
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vaccine and can be removed from isolation after the vaccine is administered and an 
appropriate sterile dry dressing is secured over the injection site. 

Participants should be provided with the Vaccinia-Prostvac Participant Instruction Sheet at 
the time of or prior to administration of Prostvac-V (APPENDIX B).
3.2.2
CV301

The CV301 vaccine regimen consists of MVA-BN-CV301 (priming vaccine) and FPV-
CV301 (booster vaccine).

MVA-BN-CV301 is administered as four subcutaneous injections (4 x 108 infectious 
units/0.5 mL each) on C1D1 and C1D15 followed by FPV-CV301 (1 x 109 infectious units/ 
0.5mL) administered on C2D1 then monthly for an additional 5 months. 
o
MVA-BN-CV301 is administered as one injection in each of the following locations: 
upper left arm, upper right arm, upper left thigh and upper right thigh (i.e., four injection 
sites per dose). Alternate subcutaneous sites may be used with approval of the principal 
investigator.
o
FPV-CV301 is administered as a subcutaneous injection preferably in the upper arm 
(i.e., one injection site per dose). An alternate subcutaneous site may be used with 
approval of the principal investigator.
3.2.3
MSB0011359C
Effective with amendment v7/29/2021, MSB0011359C will no longer be given as part of the 
treatment combination for the Biochemical Recurrence cohort (Arms 2 and 3).

MSB0011359C (anti-PDL1 TGFβ Trap) will be administered at a dose of 1200mg by IV 
infusion over 60 minutes every 2 weeks for 3 months (6 doses) starting after 4 months of 
concurrent Prostvac and CV301. Use of an in-line 0.2 or 0.22 micron filter during infusion 
administration is required.

Current experience revealed that infusion related reactions (IRRs) to MSB0011359C 
seldom occur and are generally mild to moderate in severity. Therefore, administration of 
a premedication is generally not required. 
o
If an Investigator deems it necessary to administer a premedication to a 
particular participant, an antihistamine (e.g., diphenhydramine 25-50mg  and 
acetaminophen 650mg intravenously or equivalent oral dose) is recommended 
approximately 30 - 60 minutes prior to the MSB0011359C infusion. If Grade ≥ 
2 infusion reactions are seen during the first two infusions, premedication 
should not be stopped. Premedication regimens may be adjusted based on local 
site procedures or participant-specific factors. Steroids as premedication are not 
permitted.    

Infusion-related reactions to MSB0011359C
o
Participants experiencing a grade 4 infusion related reaction will have the infusion 
stopped immediately and will not be rechallenged. 

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o
Participants experiencing a ≥ grade 2 infusion related reaction will have the infusion 
duration extended to 120 minutes for remainder of that infusion and all subsequent 
infusions. 

MSB0011359C should be administered in a setting that allows for immediate access to an 
intensive care unit or equivalent environment and administration of therapy for 
anaphylaxis, such as the ability to implement immediate resuscitation measures. Steroids 
(dexamethasone 10 mg), epinephrine (1:1,000 dilution), allergy medications (IV 
antihistamines), bronchodilators, or equivalents, and oxygen should be available for 
immediate access. 
3.2.4
Administering multiple agents

When CV301 and Prostvac are administered concurrently, it is preferable that CV301 be 
administered prior to Prostvac. When CV301 and Prostvac are administered in the same 
appendage, the vaccines must be administered a minimum of 2 inches apart. The sterile dry 
dressing to cover the Prostvac-V site should NOT overlap or cover the CV301 injection 
site. 

When CV301, Prostvac, and MSB001359C are administered concurrently, the following 
order of administration is preferred: CV301 followed by Prostvac followed by the 
MSB0011359C infusion. Effective with amendment v7/29/2021, MSB0011359C will no 
longer be given as part of the treatment combination for the Biochemical Recurrence cohort 
(Arms 2 and 3).
3.3
TREATMENT MODIFICATIONS
Effective with amendment v7/29/2021, MSB0011359C will no longer be given as part of the 
treatment combination for the Biochemical Recurrence cohort (Arms 2 and 3).
3.3.1
Dose modifications
Dose reductions or dose escalations are not permitted for Prostvac, CV301 or MSB0011359C.
3.3.2
Dosing delays 
To account for scheduling or logistical issues, the scheduled vaccine may be given ± 3 days from 
the scheduled administration date in month one of vaccine combination. Once participants are 
beyond C2D1, there will be a ±7 day window for scheduling to allow for logistical and travel 
constraints. If the vaccines or anti-PDL1 TGFβ Trap are not given within this window, it will be 
considered a missed dose and the next dose will be given at the planned administration date. If 
one agent is delayed, all agents given concurrently should also be delayed so the agents are 
administered on the same day. However, if a dose of one agent is missed for a toxicity 
unequivocally attributed to that agent, the other agents may be administered as scheduled. If for 
any reason, the immunotherapy regimen is held for >56 days, the participant will not receive 
further treatment and will be removed from the protocol.
The immunotherapy regimen must be delayed for the following drug-related AEs:

Any grade ≥ 2 drug-related AE with the following exceptions
o
Grade 2 drug-related fatigue, dermatologic AEs, and asymptomatic laboratory 
abnormalities do not require a treatment delay

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o
Grade 2 electrolyte abnormalities that can be managed with replacement therapy do 
not require a treatment delay
o
Grade 2 hypothyroidism that can be managed with replacement therapy does not 
require a treatment delay
o
Grade 2 lymphopenia or leukopenia does not require a dose delay.
o
Asymptomatic ALT/AST or amylase/lipase elevation not associated with clinical 
manifestations does not require a dose delay. Increased monitoring may be considered. 

Any grade 3 drug-related AE with the following exceptions
o
Grade 3 lymphopenia or leukopenia does not require a dose delay
o
Grade 3 asymptomatic electrolyte abnormalities that can be managed with 
replacement therapy do not require a treatment delay
o
Asymptomatic grade 3 amylase or lipase elevation not associated with clinical 
manifestations of pancreatitis does not require a dose delay. Increased monitoring is 
required. 
Criteria for resuming the immunotherapy regimen:

Participants with a required delay in treatment may resume the immunotherapy regimen 
when the drug-related AEs resolve to grade ≤ 1 or baseline with the following exceptions
o
Participants who develop grade 3 injection site reactions will have their vaccine(s) 
held until injection site reaction resolves to grade ≤ 2.
o
Participants with a grade 3 dermatologic AE may resume when their skin toxicity 
resolves to grade ≤ 2.
3.3.2.1
MSB0011359C 
Effective with amendment v7/29/2021, MSB0011359C will no longer be given as part of the 
treatment combination for the Biochemical Recurrence cohort (Arms 2 and 3).
3.4
PROTOCOL EVALUATION 
NOTE:  Also see APPENDIX C.

All participants who are deemed eligible and who sign an informed consent will be 
enrolled in this trial.

A complete history and physical examination, including ECOG performance status, will 
be done within 16 days before enrollment. 

 Laboratory Studies (See also APPENDIX C)

Serum PSA 

CBC/differential with platelet count

Serum testosterone level

Serum chemistries (Na+, K+, Cl-, CO2, glucose, BUN, creatinine, albumin, calcium, 
magnesium, phosphorus, alkaline phosphatase, ALT, AST, total bilirubin, LDH, 
CK, uric acid, total protein)

Serum amylase and lipase

Serum thyroid stimulating hormone (TSH)

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
HIV/Hepatitis B/Hepatitis C tests (within 8 weeks prior to start of enrollment)

Lymphocyte phenotyping CD3/CD4/CD8

PAP (prostatic acid phosphatase)

Urinalysis unless not feasible (i.e., participant has incontinence)
3.4.1
Radiographic assessment 
Participants will undergo scheduled radiographic assessment for metastatic disease at screening, 
prior to vaccine therapy and, at the end of study therapy, then every 6 months thereafter as long as 
participants remain in follow-up. After year 1, the PI has the discretion to perform scans q6 months 
or as clinically indicated. Radiologic studies consisting of bone scan, CT scan of chest, and CT 
scan of abdomen/pelvis will be performed at baseline. MRI may be substituted for CT scan at the 
discretion of the investigator. The window for the scans is ± 21 days,
For participants in the BCR cohort, NaF PET scans will be obtained when feasible at any point 
during the up to  4-month surveillance period and at the post treatment follow up visit. If a NaF 
PET scan was done prior to consent within 8 weeks, it will not need to be repeated during the 
surveillance period. 
3.4.1.1
Imaging with 18F-DCFPyL (Pylarify)
When feasible, participants in the BCR cohort will undergo two 18F-DCFPyL PET/CT scans: one 
in the first two months during surveillance and within 8 weeks after completing treatment. All PET 
data sets will be reviewed for quality and completeness. Participants will be instructed to maintain 
good hydration for the 24 hours prior to the 18F-DCFPyL PET/CT (recommended 1-2 liters of 
fluid, unless medically contraindicated). There are no dietary or food restrictions.
Participants will report to the appropriate imaging center on the day of their 18F-DCFPyL PET/CT 
imaging session and peripheral venous access will be obtained. 
A static whole-body PET/CT will be performed 1 hour (± 15 minutes) post injection of 18F-
DCFPyL. Only a single injection of 18F-DCFPyL is required. 
3.4.2
Assessment for immunotherapy administration 
Effective with amendment v7/29/2021, MSB0011359C will no longer be given as part of the 
treatment combination for the Biochemical Recurrence cohort (Arms 2 and 3).
Dosing and administration of CV-301, Prostvac, and MSB0011359C will be performed in the 
Clinical Center Oncology Day Hospital or on a 3rd floor oncology inpatient unit. Participants will 
be monitored with vital signs (blood pressure, heart rate, respiratory rate, temperature) prior to and 
within 1 hour after the initial vaccine treatment. On subsequent vaccine visits, participants will 
have vital signs checked prior to vaccine administration and then be monitored for 30 minutes after 
vaccine treatments (post-vaccine vital signs will not be required except for the first vaccination). 
When all three agents (CV-301, Prostvac, MSB011359C) are administered concurrently, vital 
signs will be monitored prior to vaccine administration and within 1 hour after the completion of 
the MSB011359C infusion. Documentation of any participant reported symptoms occurring 
between dosing will be included in the assessment. Laboratory assessments will be conducted as 
per APPENDIX C.

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3.5
COST AND COMPENSATION
3.5.1
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 if they 
are not covered by an insurance company. Medicines that are not part of the study treatment will 
not be provided or paid for by the NIH Clinical Center.  
3.5.2
Compensation
Participants will not be compensated on this study.
3.5.3
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. 
3.6
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 28 days (± 7 days) following the last dose of study therapy.
If a participant experiences a toxicity that can unequivocally be attributed to one agent, he may 
continue on the remaining investigational agents at the discretion of the principal investigator. 
3.6.1
Criteria for removal from protocol therapy 
Participants will be removed from treatment for the following:

Clinical or radiographic progression of disease as defined in Section 6.3.1. (Clinical 
progression includes the initiation of ADT in participants with biochemical recurrent 
prostate cancer.)

AE-related treatment delays lasting > 56 days, as defined in Section 3.3.2

Any Grade 4 toxicity that is possibly, probably or definitely related to the protocol 
treatment will require a participant to be off-treatment

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

Requirement for androgen deprivation therapy

Completion of protocol therapy 
3.6.2
Off-Study Criteria

For lead-in cohort, subjects will be taken off study at the completion of safety follow-up 
visit/period.

Participant requests to be taken off study. Reasons for withdrawal will be documented.

Requirement for androgen deprivation therapy

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
Noncompliance with protocol guidelines (participant removed at discretion of Principal 
Investigator)

Investigator decision to end the study

Death 
3.7
FOLLOW-UP EVALUATIONS
After subjects have stopped taking the study medication for any of the reasons listed in Section 
3.6.1, they will be seen at the study site, if logistically feasible, for a safety visit approximately 28 
days (+/- 14 days) after drug discontinuation. For the biochemical recurrence cohort, follow up 
will continue monthly for the first five months. For those participants who remain on study 5 
months beyond their last treatment follow-up visits could be conducted at 4-8 week intervals. 
The following assessments will be performed at the follow up safety visit:

History and Physical Examination

Serum chemistries (Na+, K+, Cl-, CO2, glucose, BUN, creatinine, albumin, calcium, 
magnesium, phosphorus, alkaline phosphatase, ALT, AST, total bilirubin, LDH, CK, uric 
acid, total protein)

Serum amylase/lipase

Serum TSH

CBC/differential with platelet count

Serum PSA level

Serum PAP

Adverse event reporting  

NaF PET scan (for Participants in the BCR cohort)
After the safety visit, if there are no unresolved grade 3 or higher AEs, we may, when feasible 
contact the participant annually to find out how they are doing and to determine survival status. If 
there are unresolved grade 3 – 4 AEs and participants cannot return for logistical reasons or refuse 
follow-up at NIH, participants may be followed by their local physician. In the latter case, we will 
obtain the physician’s medical notes and we will assess the status of the participant’s adverse 
events based on the physician’s reports.
Any scans performed outside of the study site will also be obtained when possible. 
4
CONCOMITANT MEDICATIONS
4.1
EXCLUDED MEDICATIONS

Concurrent hormonal therapy, anticancer treatment with chemotherapy, radiation therapy, 
major surgical procedures for prostate cancer, anti-cancer radionuclides and non-protocol-
related immunotherapy will not be permitted. 

Systemic corticosteroids (daily or every other day for continued use > 14 days) will not be 
permitted. Participants requiring systemic steroids for treatment of immune-related AEs 
may remain on-study at the discretion of the investigator. Corticosteroids with minimal 

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systemic absorption (e.g., topical and inhaled steroids) are allowed. Ophthalmologic 
steroids will not be permitted within 3 days prior to and within one month after receiving 
the Prostvac-V injection. 

Vaccination with live vaccines other than the investigational agents is prohibited (e.g., 
Zostavax). Administration of inactivated vaccines is allowed (e.g., inactivated influenza 
vaccine). Locally approved COVID vaccines are permitted. 
4.2
SUPPORTIVE CARE
4.2.1
General supportive care
For the administration of Prostvac and CV301 , antiemetics, 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, analgesics, general medical therapy, etc., will be 
delivered as required. 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.
4.2.2
Antibiotics
Antibiotics will be delivered as required. Co-administration of protocol treatments can be done at 
the discretion of the investigator. Prophylactic antibiotics (e.g., prevention of urinary tract 
infections) may be allowed at the discretion of the investigator. 
4.2.3
Infusion-Related Reactions 
See section 3.2.3. 
4.2.4
Immune-Related Adverse Events (irAEs)
Immune-related AEs (irAEs) require early recognition and prompt intervention. If an irAE is 
suspected, all investigational agents must be held. See management algorithms in Error! 
Reference source not found..
4.3
TREATMENT OF VACCINIA VACCINATION COMPLICATION
4.3.1
Vaccinia Immune Globulin:
First-line treatment of some of the complications of vaccinia caused by dissemination of vaccinia 
virus (severe cases of inadvertent inoculation involving extensive lesions or if comorbid conditions 
exist, severe cases of generalized vaccinia in participants that are systemically ill and whose 

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condition might be toxic or who have serious underlying immunosuppressive illnesses, eczema 
vaccinatum, and progressive vaccinia) is with VIG.
VIG is contraindicated, however, for the treatment of isolated vaccinial keratitis. VIG is a sterile 
solution of the immunoglobulin fraction of pooled plasma from individuals inoculated with 
vaccinia vaccine. VIG is an investigational agent available through the CDC’s Strategic National 
Pharmaceutical Stockpile under an IND protocol by contacting the CDC’s Smallpox Vaccine 
Adverse Events Clinician Information Line at 1-877-554-4625. Upon receipt of a call from a 
participant or upon direct observation of a participant or contact who manifests signs and 
symptoms of any of the above conditions, the investigator should place a call to the CDC as soon 
as possible:
1) to initiate review of the clinical case, 
2) to seek consultation on the appropriateness of VIG therapy, 
3) to determine the appropriate VIG dose and dosing method for administration, if VIG
therapy is required, and 
4) to determine how to access and have the appropriate doses of VIG delivered.
Early institution of VIG therapy is advised following recognition of clinical symptoms compatible 
with some vaccinia complications (eczema vaccinatum, severe generalized vaccinia, progressive 
vaccinia, and some cases of inadvertent inoculation). The effectiveness of VIG therapy appears to 
be time dependent. VIG has not proven to be of benefit in the treatment of post-vaccinial 
encephalitis, and is contraindicated for treatment of isolated vaccinial keratitis due to the increased 
risk of corneal scarring.
An intravenous formulation of VIG is available through the CDC, which has a lower level of 
aggregated protein, allowing it to be used by either the IM or IV route. This formulation will most 
likely be preferred for administration and investigators will be instructed by the CDC regarding 
appropriate dosing and method of administration based on formulation and availability. There is 
no guarantee that VIG will successfully treat complications. At present, there are no other anti-
viral therapies of proven benefit for the treatment of vaccinia-related complications.
4.3.2
Cidofovir (Vistide®, Gilead Sciences):
Cidofovir is an FDA-approved antiviral drug for the treatment of CMV retinitis among participants 
with AIDS. Cell-based in vitro studies and animal model studies have demonstrated antiviral 
activity of this agent against certain orthopoxviruses. Currently, efficacy in the treatment of 
vaccinia-related complications in humans is unknown. According to the CDC, “VIG is 
recommended as first line of therapy. Cidofovir may be considered as a secondary treatment, and 
will only be released by the CDC after all inventories of VIG have been exhausted, after a 
participant fails to improve with VIG treatment, or as a last effort for a participant who is otherwise 
near death.” [Medical Management of Smallpox (Vaccinia) Vaccine Adverse Reactions: Vaccinia
Immune Globulin and Cidofovir. Last updated February 11, 2003. Available at:
https://www.cdc.gov/smallpox/clinicians/vaccine-medical-management6.html].

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5
BIOSPECIMEN COLLECTION
5.1
CORRELATIVE STUDIES FOR RESEARCH
5.1.1
Immunologic Parameters

Antibodies to PSA, vaccinia, fowlpox may be tested

Leukocyte CD3, CD4, CD8 subsets; CD4:CD8 ratio will be drawn according to the 
Research Bloods schedule in the study calendar (see APPENDIX C) while the 
participant remains on trial.

The results of the HIV antibody need to be available before treatment to determine 
eligibility.

Additional studies will include but are not limited to quantitative and qualitative 
assessments of regulatory T-cells, Natural Killers cells, Myeloid Derived Suppressor 
Cells, anti-glycan antibodies and Naïve T-cell/new thymic emigrants. 

Assessment of levels of cytokines.

Immunologic studies will be repeated more frequently if clinically indicated, and any 
abnormalities potentially related to treatment will be followed until they have resolved, 
or have been determined not to be treatment-related.
5.1.2
Summary of Sample Collection
Sample
Collection Details*
Collection point    
Location of specimen 
analysis
Blood sample 
for PBMC: 
Immune 
Monitoring
6 x 10 mL green top sodium 
heparin tubes (also listed in 
APPENDIX D)
See study calendar in  
APPENDIX C
Clinical Services Program
NCI Frederick Research 
and Development Center
Blood sample 
for serum:  
Immune 
Monitoring
2 x 8 mL SST tubes (also 
listed in APPENDIX D)
See study calendar in 
APPENDIX C
Clinical Services Program
NCI Frederick Research 
and Development Center
Stool 
Samples
Stool Collection Tubes
See study calendar in 
APPENDIX C
Figg Lab
*Tubes/media may be adjusted at the time of collection based upon materials available. The amount of 
blood that may be drawn from adult participants for research purposes shall not exceed 10.5 mL/kg or 
550 mL, whichever is smaller, over any eight-week period.
NOTE:  Portions of all samples may be banked for future research analyses; prospective consent will be 
obtained during the informed consent process. 

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5.1.3
Immunologic Assays 
5.1.3.1
CD4 T Cell Proliferation Assay
It is planned that all participants will undergo exploratory analysis of the ability to detect CD4-
positive responses using a whole-protein PSA assay, as well as a peptide mix with 63 different 15-
mer peptides by ELISPOT and/or ELISA. 
5.1.3.2
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. 
5.1.3.3
Flow cytometry analysis of thymic emigrant
To determine recent thymic emigrants, flow cytometry analysis will be performed on peripheral 
blood mononuclear cells. Cells will be resuspended in staining buffer (phosphate-buffered saline 
containing 3% fetal bovine serum) and stained for 30 minutes at 4°C with the combination of 
following antibodies: APC-H7-conjugated anti-CD4, PE-CY7-conjugated anti-CD3; FITC-
conjugated CD45RA, PE-conjugated CD31, PerCP-CY5.5-conjugated Ki-67, AF-700-
conjugated-CD197, V450-conjugated CD8, APC-conjugated CD103, V500-conjugated CD27 all 
purchased from BD Pharmingen, San Diego, CA). After that, FoxP3 intra-cellular staining will be 
performed on the cells stained with anti-CD4 and anti-CD25. They will be fixed and 
permeabilyzed using a fix/perm kit (eBioscience, San Diego, CA) according to the manufacturer’s 
manual, and will be labeled with FITC-conjugated anti-Foxp3 antibody (236A/E7 clone) or its 
isotype control antibody (eBioscience). Flow cytometry will be performed on a Becton Dickinson 
LSRII (BD Biosciences) device.
5.1.3.4
Natural Killer (NK) Cells
The number and phenotype of NK cells will be determined by phenotypic analysis of PMBCs 
stained for CD56, CD3, CD8, and CD16 by flow cytometry.
5.1.3.5
Immune Subsets
Subsets of immune cells will also be followed in response to treatment.
5.1.3.6
Regulatory T Cells
Regulatory T cells have been shown to inhibit the activation and function of T cells that participate 
in antigen-specific immune responses. Higher levels of regulatory T-cells have been reported in 
the peripheral blood mononuclear cells of participants with several types of tumors. The number 
and phenotype of regulatory T-cells in peripheral blood mononuclear cells from participants in this 
study will be determined by 7-color flow cytometry analysis. Cells will be resuspended in staining 
buffer (phosphate-buffered saline containing 3% fetal bovine serum) and stained for CD4, CD25, 
CD127, FoxP3, CTLA-4, CD45RA, and CD8. The ratios between regulatory T-cells and CD4 
effector cells and the ratios between regulatory T-cells and CD8 Effector cells will also be 
analyzed.
5.1.4
Stool (optional)
Participants will be asked to provide stool in order to evaluate changes in microbiome of 
gastrointestinal tract and correlate to changes in treatment, PSA and immune cell subpopulations.

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biological specimens in support of NIH clinical studies. All specimens are stored in secure, 
limited-access facilities with sufficient security, backup, and emergency support capability and 
monitoring to ensure long-term integrity of the specimens for research. 
The subcontractor’s role is limited to clinical research databases and repositories containing 
participant specimens. It does not conduct or have 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 NCI Frederick Central Repository will accept only coded, linked samples and sample 
information. Protected information will not be 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 to 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, 3 types of user access levels, and 36 user permissions 
(levels of access) that can be set to control access to the system functions. BSI 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 bar-coded 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.
Samples will be used for research analysis, including immunologic monitoring as outlined in 
Section 5.1. All specimens for analysis will be requested from Leidos Biomedical, Inc. and will be 
delivered by Leidos Biomedical, Inc. couriers to the Laboratory of Tumor Immunology and 
Biology.
5.3.2
Stool Samples (Blood Processing Core/Figg Laboratory)
All samples sent to 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.
Following completion of this study, samples will remain in storage as detailed above. Access to 
these samples will only be granted following IRB approval of an additional protocol, granting the 
rights to use the material. 
If, at any time, a participant withdraws from the study and does not wish for their existing samples 
to be utilized, the individual must provide a written request. Following receipt of this request, the 
samples will be destroyed (or returned to the participant, if so requested), and reported as such to 
the IRB. The PI will record any loss or unanticipated destruction of samples as a deviation. 
Reporting will be per the requirements of section 7.2.
Sample barcodes are linked to participant demographics and limited clinical information. This 
information will only be provided to investigators listed on this protocol, via registered use of the 
Labmatrix. It is critical that the sample remains linked to participant information such as race, age, 
dates of diagnosis and death, and histological information about the tumor, in order to correlate 
genotype with these variables.
5.3.3
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.
5.4
SAMPLES FOR GENETIC/GENOMIC ANALYSIS
5.4.1
Description of the scope of genetic/genomic analysis
Genomic sequence characterization is increasingly important in understanding tumor biology, 
host/microbial interactions, and skin conditions. The current study proposes to perform DNA 
sequencing on biologic samples, including stool samples – although the likelihood of incidental 
human genomic findings in stool sampling is expected to be very low/rare, the possibility for this 
is included in an abundance of caution.
The technology platforms used to interrogate genomic structure and function are dynamic and 
the specific methodology, e.g. DNA/RNA sequencing, DNA copy number analysis, DNA 

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methylation analysis, employed will be determined at the time that samples are ready for 
analysis.
5.4.1
Description of how privacy and confidentiality of medical information/biological 
specimens will be maximized
Confidentiality for genetic samples will be maintained as described (Section 5.3). In addition, a 
Certificate of Confidentiality has been obtained for this study.
5.4.2
Management of Results 
Subjects will be contacted if a clinically actionable gene variant is discovered. Clinically 
actionable findings for the purpose of this study are defined as disorders appearing in the American 
College of Medical Genetics and Genomics recommendations for the return of incidental findings 
that is current at the time of primary analysis. Subjects will be contacted at this time with a request 
to provide a sample to be sent to a CLIA certified laboratory. If the research findings are verified 
in the CLIA certified lab, the subject will be offered the opportunity to come to NIH (at our 
expense) to have genetic education and counseling to explain this result. If the subject does not 
want to come to NIH, a referral to a local genetic healthcare provider will be provided (at their 
expense).
This is the only time during the course of the study that incidental findings will be returned. No 
interrogations regarding clinically actionable findings will be made after the primary analysis.
6
DATA COLLECTION AND EVALUATION
6.1
DATA COLLECTION

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 a 21 CR-Part 11-compliant data capture system provided by the 
NCI CCR. 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.
The PI will be responsible for overseeing entry of data into a 21 CR-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.

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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 28 days after last dose of 
study treatment.
An abnormal laboratory value will be considered an AE 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

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. 
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).

Coded, linked or identified 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. Insert name or names:__clinicaltrials.gov.

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? 

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
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 
Response assessments will be performed per the schedules in the study calendars in APPENDIX 
C.
6.3.1
Disease Progression

PSA will not be used to measure progression of disease. Participants will be offered 
androgen deprivation therapy (ADT) as clinically indicated. Starting ADT will be done at 
the discretion of the investigator, although this will result in treatment termination and 
removal from the clinical trial. Participants may go on ADT at any time, although this may 
result in removal from the trial. PSA will be used to calculate PSA kinetics/tumor growth 
kinetics.

Development of a new bone lesion on bone scan.

Development of a soft tissue mass, identified on CT scan or physical exam, consistent with 
metastatic prostate cancer. If identified on physical exam, the lesion may be biopsied to 
confirm the presence of prostate cancer.

Development of urethral, ureteral, or spinal cord obstruction secondary to tumor.

Development of cytologically positive pleural effusion or lymphangitic spread in the lungs.

Symptoms which in the opinion of the investigator are consistent with clinical progression.
6.3.2
 Intra-study PSA response 
Participants will be monitored for PSA kinetics once enrolled. Participants who have an intra study 
decline in PSA will be evaluated for PSA response. Such responses would be defined by an intra-
study apex PSA, which will be established by determining the maximum PSA value that can be 
validated by another intra-study PSA value within 10%. (The goal here is to not capture lab 
variations which may be seen as isolated PSA spikes (i.e. greater than 10%) in the context of 
participant follow-up. Responders will then be evaluated using confirmed PSA declines (at least 2 
consecutive values after the intra-study apex) of at least 30%. Participants with intra-study PSA 
declines greater than 50% will also be identified.
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#ctc_40). 

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7
NIH REPORTING/DATA AND SAFETY MONITORIING 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. 
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 Vaccine 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 Vaccine, but are not fatal or life-
threatening, much 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 information 
described below shall be submitted. 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.

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7.4.2.1
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. 
7.4.2.2
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

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 Principal Investigator, lead associate investigator and the research nurse will meet weekly at 
each clinic to review all adverse events for each subject in this trial. Unexpected adverse events 
and/or serious adverse events will be reported to the NIH Intramural Institutional Review Board 
(IRB) and sponsor/FDA as outlined above. If trends are noted and/or risks warrant it, accrual will 
be interrupted, dose levels expanded and/or the protocol and/or consent will be modified 
accordingly.

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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 
7.2.1 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 patient 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)).
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 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 or research subject 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.

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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.
•
Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event.
8.1.6
Adverse Events of Special Interest
Adverse events of special interest (AESIs) are serious or nonserious AEs that are of clinical 
interest and should be closely followed. 
AESIs include the following:

Infusion-related reactions including immediate hypersensitivity

Immune-related adverse events

TGFβ inhibition mediated skin reactions

Anemia

Bleeding AEs
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.

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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 
CMP updates.
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 

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Protocol Deviation Tracking System (PDTS) for deviation reporting to the Sponsor and as 
applicable per institutional and IRB guidance.
10 STATISTICAL CONSIDERATIONS   
10.1 STUDY OBJECTIVES
10.1.1 Primary
The primary objective is to determine if the combination immunotherapy can result in a confirmed 
30% decline in PSA in a modest fraction of participants enrolled
10.1.2 Secondary 
The secondary objectives are to:

Assess the safety of the combination immunotherapy regimen

Test for changes in PSA kinetics after treatment with the combination immunotherapy, 
relative to baseline PSA kinetics
10.2 SAMPLE SIZE DETERMINATION
The trial will begin with a 6-participant safety lead-in. Following determination of safety, the 
primary objective of this study is to determine if the use of two vaccines combined with an immune 
checkpoint inhibitor can be associated with a modestly high fraction of participants who can 
experience at least a 30% decline from their maximum PSA anytime during treatment to the 
minimum value during treatment.
Safety data for the 6-participant lead-in will be shared with IRB before accrual commences in the 
biochemical recurrence cohort. Beyond safety, this cohort will just be evaluated in a descriptive 
manner.
Because participants may be receiving the proposed treatment and be followed for PSA changes 
over an extended period, and the evaluation of the endpoint may take place at any point during this 
potentially long period, the trial will use a single stage design for evaluating efficacy. With 25 
evaluable participants who do not receive M7824, there would be 82% power to rule out 15% and 
be consistent with 35% who may be able to experience a 30% decline in PSA, using an exact 
binomial test with a 0.10 one-sided significance level. As an illustration, if 25 evaluable 
participants are enrolled, 7 of 25 (28.0%) who experience at least a 30% PSA change from the 
maximum level would have an associated one-sided lower 90% confidence interval bound of 
16.3%, and simultaneously an associated one-sided upper 90% confidence interval bound of 
42.6%. These demonstrate that 7/25 would be a desirable result based on the parameters selected.
With the amendment v7/29/2021, the initial 6 participants who received M7824 will be evaluated 
separately in exploratory fashion and will not be included among the 25 main participants.
In addition, because a trial of participants with similar eligibility (16-C-0035) may result in 
approximately 36 participants who are undergoing surveillance for 6 months, this other trial may 
serve as a control for a secondary evaluation. The changes in PSA on this trial during the initial 6 
months can be compared to the changes in PSA during the initial 6 months on the surveillance arm 
of the control trial as a secondary evaluation. With 25 evaluable participants on this trial and 36 
evaluable participants on the other trial, there would be 81% power to identify a difference between 
35% of participants on this trial with a 30% PSA maximum change and 10% of participants on the 

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control trial with a 30% PSA maximum change with a 0.10 one-sided significance level Fisher’s 
exact test.
It is anticipated that 1-2 participants per month can be enrolled onto this trial; thus, it expected that 
accrual of 37 evaluable participants (6 for the lead-in, 6 who received M7824 as part of the initial 
investigation for PSA response and 25 homogeneously treated participants to evaluate PSA 
response) can be completed within 1 to 2 years. To allow for a small number of inevaluable 
participants, the accrual ceiling will be set at 40 participants.
10.3 POPULATIONS FOR ANALYSIS
Modified intention to treat: all participants who receive at least one dose of the two vaccines and 
the immune checkpoint inhibitor will be included in the statistical analyses performed. The lead-
in cohort will be evaluated separately in a descriptive fashion and for safety primarily.
10.4 STATISTICAL ANALYSES
10.4.1 General approach
Following a safety determination in 6 participants meeting separate eligibility requirements, the 
PSA decline measured from the maximal value obtained while on study to the minimum value 
obtained while on study will be obtained for each patient, and will be considered a success if the 
maximal PSA decline is 30% or more. The fraction of evaluable participants who experience at 
least a 30% decline will be determined. Quantitative parameters will be tested for changes over 
time using paired tests.
10.4.2 Analysis of the primary efficacy endpoints
The fraction of evaluable participants who experience at least a 30% decline from the maximum 
to the minimum PSA value while on study will be determined. A two-tailed 80% and 95% 
confidence interval will be formed around the fraction of participants with 30% decline to interpret 
the fraction who experience a decline of that magnitude. This primary analysis will not include the 
6 patients who received M7824 before it was removed from future use in this study.
10.4.3 Analysis of the secondary efficacy endpoints
A 6-participant phase I lead in cohort in advanced (castration resistant prostate cancer) participants 
will be conducted to determine safety. Once all 6 participants have completed a 6-week toxicity 
assessment period the toxicity data will be shared with IRB. If the IRB approves accrual to evaluate 
PSA response, then the trial will move forward with the planned study in biochemical recurrence. 
The participants in the safety cohort will only be analyzed for safety and toxicity, and not PSA 
changes.
Safety of the combination regimen will be assessed by reporting the grade of adverse events noted 
in each participant, and reporting the fraction with grade 3 and grade 4 adverse events. 
To test for changes in PSA kinetics after treatment with the combination immunotherapy relative 
to baseline PSA kinetics, the changes in PSA over defined intervals (e.g., 1 to 3 months or similar) 
after administering the vaccines as well as after administering the checkpoint inhibitor will be 
determined by calculating the slope of the PSA change over time. Then, the slopes obtained after 
administering the vaccines alone or in combination with the checkpoint inhibitor will be tested vs. 
the slopes obtained prior to administering the vaccines using a two-tailed 0.05 significance level 
paired t-test or Wilcoxon signed rank test (if the paired differences are not normally distributed). 

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In addition, because a trial of participants with similar eligibility (16-C-0035) may result in 
approximately 36 participants who are undergoing surveillance for 6 months, this other trial may 
serve as a control for a secondary evaluation. The changes in PSA on this trial during the initial 6 
months can be compared to the changes in PSA during the initial 6 months on the surveillance arm 
of the control trial as a secondary evaluation. The fractions of participants who experience a 30% 
PSA maximum change on the present study as well as the approximately 36 participants on 16-C-
0035 who underwent surveillance will be compared using a one-sided significance level Fisher’s 
exact test.
Please note: As of December 21, 2018, the 6 participants in the lead in cohort have completed the 
6-week toxicity assessment period and no SAEs were seen. Per the protocol design participants 
will now be enrolled to the BCR cohort. The toxicity assessment of participants in the lead-in 
cohort is described in section 1.2.13.
10.4.4 Safety Analyses
Safety of the combination regimen will be assessed by reporting the grade of adverse events noted 
in each participant, and reporting the fraction with grade 3 and grade 4 adverse events.
10.4.5 Baseline Descriptive Statistics
Demographic and clinical characteristics of all participants will be reported.
10.4.6 Planned interim analyses
None will be performed.
10.4.7 Subgroup analyses
None will be performed, except to consider exploratory evaluations based on the use of M7824
10.4.8 Tabulation of individual participant data
None will be provided.
10.4.9 Exploratory analyses
The study will aim to evaluate immune changes after treatment with 2 vaccines for 4 months.
Many immune parameters (See Section 5.1) will be obtained at baseline and at several time points 
beginning at the time the two vaccines are first administered. The changes in the immune 
parameters after treatment with 2 vaccines for 4 months will be determined and tested for statistical 
significance using a two-tailed 0.05 significance level paired t-test or Wilcoxon signed rank test 
(if the paired differences are not normally distributed). Since there are potentially a moderately 
large number of these tests, they will be reported as being of secondary intent and presented 
without adjustment for multiple comparisons but in the context of the number of such tests 
performed.
Willing participants will continue to be followed after cycle 7 until progression or ADT is 
clinically indicated.
Patients in cohort 2 who received M7824 as part of their therapy will have their PSA response 
evaluated in a descriptive fashion, and may be compared informally to those from the remaining 
patients who do not receive M7824.

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10.4.10
 Halting Rules
If either of the below occurs, the Principal Investigator will halt accrual to the trial and will discuss 
with the NIH Intramural Institutional Review Board (IRB) whether any changes need to be made 
to the protocol.

One occurrence of grade 5 toxicity by the NCI-CTCAE version 4.0 attributable to the 
treatment regimen.

Two occurrences of grade 4 toxicity by the NCI-CTCAE version 4.0 attributable to the 
treatment regimen.

More than two episodes of grade 3 colitis
This will apply to both cohorts-the phase I lead-in cohort and the main cohort of biochemical 
recurrence.
11 COLLABORATIVE AGREEMENTS 
11.1 COLLABORATIVE RESEARCH AND DEVELOPMENT AGREEMENT (CRADA) 
This study is conducted under a Collaborative Research and Development Agreement (CRADA) 
12 HUMAN SUBJECT PROTECTIONS 
12.1 RATIONALE FOR SUBJECT SELECTION
12.1.1 Selection Based on Gender, Ethnicity, and Race 
Subjects from all racial/ethnic groups are eligible for this study if they meet the eligibility criteria. 
To date, there is no information that suggests that differences in drug metabolism or disease 
response would be expected in one group compared with another. Efforts will be made to extend 
accrual to a representative population, but in this preliminary study, a balance must be struck 
between participant safety considerations and limitations on the number of individuals exposed to 
potentially toxic and/or ineffective treatments on one hand and the need to explore ethnic aspects 
of clinical research on the other hand. If differences in outcome that correlate with ethnic identity 
are noted, accrual may be expanded or a follow-up study may be written to investigate those 
differences more fully. Women are not eligible for this study as this disease occurs only in men.
12.1.2 Justification for Exclusions
Due to impaired cellular immunity with the concomitant increased risk of serious side effects from 
vaccinations with infectious agents, the Centers for Disease Control and Prevention recommends that 
HIV infected participants be excluded, in addition, participants with chronic hepatitis infection, including 
B and C, because of potential immune impairment.
12.2 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.

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12.3 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 could become 
decisionally impaired. For this reason and because there is a prospect of direct benefit from 
research participation (section 12.4), all subjects 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 12.6.1 for consent procedure.
12.4 EVALUATION OF BENEFITS/RISKS/DISCOMFORTS
There is no standard therapy for participants with prostate cancer and rising PSA following local 
definitive therapy. Potential risks of Prostvac, CV-301 and MSB0011359C in this patient 
population include the range of side effects outlined in Section 14 and Section 1.2. Prostvac has 
been well tolerated in previous large trials. CV-301 and MSB001359C have demonstrated 
acceptable adverse events in phase I dose escalation trials. 
Effective with amendment v7/29/2021, MSB0011359C will no longer be given as part of the 
treatment combination for the Biochemical Recurrence cohort (Arms 2 and 3). Prostvac has been 
well tolerated in previous large trials. CV-301 has demonstrated acceptable adverse events in phase 
1 dose escalation trials. 
12.4.1 Risks associated with blood sampling
Side effects of blood draws include pain and bruising in the area where the needle is inserted, 
lightheadedness, and rarely, fainting. Participants in the CRPC lead-in cohort will have 
approximately the following amounts of blood collected for routine laboratory testing: 2 ½ 
tablespoons during screening, 3 tablespoons at the start of each cycle, day 15 of cycles 1 – 3, end 
of treatment and safety visit. Participants in the biochemical recurrence portion will have 
approximately the following amounts of blood collected for routine laboratory testing: 2.5 
tablespoons during screening, 3 tablespoons during baseline, day 1 of each cycle, on day 15 of 
cycles 1, 5, 6, and 7, end of treatment and during follow up. We will collect approximately 5 
tablespoons of blood for research at baseline, and the start of cycle 1, cycle 2, cycle 5, end of 
treatment and during follow up. Over a two-week period, approximately 6 ½ tablespoons of blood 
will be collected in the lead in portion and approximately 9 tablespoons of blood will be collected 
in the biochemical recurrence portion.
12.4.2 Risks of genetic research
The planned genetic research on biospecimens are non-physical and include the following:  

Risk of receiving unwanted information:  Anxiety and stress may arise as a result of the 
anticipation that unwanted information regarding disease related DNA sequencing or 
disease tendencies, or misattributed paternity. Participants will be clearly informed that the 
data related to DNA sequencing and genetic analysis is coded, investigational and will not 
be shared with participants, family members or health care providers, unless subjects or 

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parents/guardians choose to be contacted if a clinically actionable gene variant is discovered 
(Section 5.4.2).

Risk related to possibility that information may be released:  This includes the risk that data 
related to genotype, DNA sequencing or risk for disease tendency or trait can be released to 
members of the public, insurers, employers, or law enforcement agencies.

Risk to family or relatives:  Family members or relatives may or may not want to be aware 
of familial tendencies or genetic risks of disease which may cause anxiety about possible 
future health problems.
12.4.3 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.
12.4.4 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 (APPENDIX C), and must have a local physician to provide long-term care 
and monitoring for complications. 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.
12.4.5 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 RISKS/BENEFITS ANALYSIS
This study involves clinical research with an experimental combination immunotherapy designed 
to generate an immune response against prostate cancer. Participants will undergo multiple 
vaccinations with Prostvac and CV-301. Participants in Arm 1 and Arm 2 also received multiple 
infusions of MSB001359C until that drug was removed from the study as of amendment 
v07/29/2021 due to immune related adverse events observed with M7824 in this and other studies. 
Side effects of the immunotherapy are outlined elsewhere (see Section 14 and Section 1.2). 
Potential risks are applicable for all participants on study, including those that may become 
cognitively impaired while on study. Whether the combination immunotherapy regimen will have 
any clinical effect is unknown; therefore, benefit cannot be promised, nor the chance of benefit 
accurately predicted. Potential benefits for all participants on study, including those that may 

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become cognitively impaired while on study, could include shrinking of tumor or lessening of 
symptoms, such as pain, that are caused by the cancer. Participation in this study is voluntary, and 
refusal will not result in penalty or loss of benefit to which the participant is otherwise entitled. 
We believe that genetic analyses of study samples will pose no more than minimal risk to the 
subjects or their family members, as the results will not be reported with any identifiable 
information. The codes linking the subject with samples will be maintained by the principal 
investigator and research coordinator in a locked electronic data base, with access only to the PI 
and research coordinators.
Participation may be discontinued at any time without penalty, and the participant will be 
encouraged to discuss any concerns or questions.
12.5.1 Risks related to Imaging
CT scans, Tc-99 scans and PET scans often use a contrast agent. There is a small risk of having a 
reaction to the contrast and most often include nausea, pain in the vein where the contrast is given, 
headache, metallic and/ or bitter taste in the mouth and a warm, flushing feeling. Rarely, some 
people have more severe allergic reactions to the contrast which may include skins rashes, 
shortness of breath, wheezing or low blood pressure.
There is a small risk of having a reaction to the contrast agent 18F-DCFPyL (Pylarify) and most 
often include headache, metallic and/or bitter taste in the mouth, and fatigue. Participants may 
have discomfort from lying on a hard surface for about 2 hours during the scans, infection at the 
IV site, or leaking of the contrast into the skin and tissue around the IV. Rarely, some people may 
have an allergic reaction to the contrast. 
12.5.2 Risks from Radiation Exposure 
The procedures for performing the CT scans (chest/ abdomen/ pelvis), Tc-99 whole body 
scintigraphy scans, 18F NaF PET/CT scans, and two 18F-DCFPyL PET scans will follow clinical 
policies, no special procedures apply to these additional assessments for research purposes. In 
summary, subjects may receive additional radiation exposure in one year from up to three (3) CT 
scans of the chest, abdomen, and pelvis, three (3) Tc-99 whole body scintigraphy scans and two 
(2) 18F NaF PET/CT scans. 
The total additional radiation dose for research purposes will be approximately 7.2 rem in one 
year. 
12.5.3 Risks from other study procedures
There are minimal risks associated with electrocardiogram as this is a relatively safe procedure.
Questionnaires may contain questions that are sensitive in nature. The participants are asked to 
only answer questions they are comfortable with.
12.6 CONSENT PROCESS AND DOCUMENTATION
The informed consent document will be provided as a physical or electronic document to the 
participant or consent designee(s) as applicable 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 

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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 as described below. 
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 participant 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.
12.6.1 Consent Process for Adults Who Lack Capacity to Consent to Research Participation
For participants addressed in section 12.3, an LAR will be identified consistent with Policy 403 
and informed consent obtained from the LAR, as described in Section 12.6. 
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, 
investigator, the Investigational New Drug (IND) 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:

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
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
The 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 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 NCI 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. 
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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 each 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 NIH.
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  
14.1.1 Background
Other Names: PROSTVAC-F/TRICOM; PROSTVAC-F
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) 
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the inserted transgenes to stimulate both humoral and cellular immunity, but cannot replicate in 
non-avian species, making systemic infections unlikely. 
14.1.2 How Supplied 
Recombinant Fowlpox-PSA(L155)/TRICOM will be supplied by the manufacturer, Bavarian 
Nordic, Inc. 
Recombinant Fowlpox-PSA(L155)/TRICOM is supplied 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.
14.1.3 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.
14.1.4 Storage 
Store intact vials of Recombinant Fowlpox-PSA(L155)/TRICOM at –70oC or colder. 
14.1.5 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.
14.1.6 Route of Administration 
Recombinant Fowlpox-PSA(L155)/TRICOM is administered by subcutaneous injection 
preferably in the upper thigh. Alternate subcutaneous sites may be used with approval of the 
principle investigator. 
14.1.7 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 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 
1. Strictly adhere to standard microbiological practices and techniques.
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3. Use appropriate infection control measures (e.g., thorough hand washing) after handling any 
materials.
4. Institute and follow policies for safe handling of sharps.
5. 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]. 
6. 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. 
7. 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.
8. 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.
9. Transport the agent from the freezer to the work area in leak proof bag. 
10. 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.
11. 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.
12. 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.
13. Incinerate waste according to institutional policy and according to local, state, and federal 
regulations.
14. Handle accidental spills similarly to antineoplastic spills and/or according to institutional 
policy:

Prevent others from entering the area and allow aerosols time to settle (approximately 10 
minutes). 

Use protective apparel, eyewear, mask, and gloves. 

Cover spills with disposable absorbent towels. 

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
Decontaminate the area with 10% bleach solution, or other appropriate disinfectant suitable 
for decontamination, allowing approximately a 20-minute contact time. 

Dispose of all waste as biohazardous waste and incinerate according to institutional policy 
and according to local, state, and federal regulations. 
15. Immediately report spills and accidents resulting in overt exposure to recombinant DNA 
molecules to the site’s Institutional Biosafety Committee (IBC) by the site’s PI. Provide 
medical evaluation, surveillance, and treatment as appropriate and maintain written records of 
the event.
For more information about biohazard risk group classification and biohazard safety levels see:

NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH 
Guidelines). 
See 
current 
version 
at: 
http://osp.od.nih.gov/sites/default/files/NIH_Guidelines.html

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 
Participant 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 participants to avoid direct contact of the injection site with susceptible individuals (e.g.; 
those who may be immunocompromised by disease or therapy). Instruct participants to avoid 
fathering a child for at least 4 months following therapy completion with the recombinant 
vaccine. Instruct participants 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), 
participants with a history of allergy to eggs or egg products should not receive the vaccine.
14.2 RECOMBINANT VACCINIA-PSA(L155)/TRICOM 
14.2.1 Background
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 

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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 humoral and cellular 
immune response. Vaccinia virus is replication competent in mammalian cells, making systemic 
infections possible.
14.2.2 How Supplied 
Recombinant Vaccinia-PSA(L155)/TRICOM will be supplied by the manufacturer, Bavarian 
Nordic, Inc. 
Recombinant Vaccinia-PSA(L155)/TRICOM is supplied 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. 
14.2.3 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.
14.2.4 Storage 
Store intact vials of Recombinant Vaccinia-PSA(L155)/TRICOM at –70oC or colder.
14.2.5 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.
14.2.6 Route of Administration 
Recombinant Vaccinia-PSA(L155)/TRICOM is administered by subcutaneous injection 
preferably in the upper thigh. Alternate subcutaneous sites may be used with approval of the 
Principal Investigator.  
14.2.7 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 

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1. Prepare a biosafety manual which advises personnel of special hazards and specific 
instructions on practices and procedures.
2. 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.
3. 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.
4. Strictly adhere to standard microbiological practices and techniques.
5. Limit/restrict access to preparation areas while dose preparation is in progress.
6. Use appropriate infection control measures (e.g., thorough hand washing) after handling any 
materials.
7. 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.
8. 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]. 
9. Perform all procedures carefully to minimize aerosol creation.
10. 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. 
11. 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.
12. Place an empty biohazard sharps container in the biosafety cabinet to dispose of all waste 
generated.
13. Transport the agent from the freezer to the work area in leak proof bag. 
14. 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.
15. 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 
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16. 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.
17. 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.
18. Handle accidental spills similarly to antineoplastic spills and/or according to institutional 
policy:
a.
Prevent others from entering the area and allow aerosols time to settle (approximately 10 
minutes). 
b. Use protective apparel, eyewear, mask, and gloves. 
c.
Cover spills with disposable absorbent towels. 
d. Decontaminate the area with 10% bleach solution, or other appropriate disinfectant suitable 
for decontamination, allowing approximately a 20-minute contact time. 
e.
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. 
19. Immediately report spills and accidents resulting in overt exposure to recombinant DNA 
molecules to the site’s Institutional Biosafety Committee (IBC) by site’s PI. Provide medical 
evaluation, surveillance, and treatment as appropriate and maintain written records of the 
event.
For more information about biohazard risk group classification and biohazard safety levels: 

NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH 
Guidelines). 
See 
current 
version 
at: 
http://osp.od.nih.gov/sites/default/files/NIH_Guidelines.html

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 

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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): 

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, or other open rashes or wounds), until the condition resolves.

are pregnant or intend to become pregnant within 4 weeks of vaccination or are breast-feeding.

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 vaccine:  

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.

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 participants, or the participant’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 Participant Care Implications, Contraindications and Potential 
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 Participant Care Implications and Contraindications
Cover vaccination sites with a sterile dry dressing (e.g., Telfa pad). Instruct participants on proper 
hand-hygiene, sterile dressing care, bathing, laundering of clothing, etc. Treat participant 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. Participants 
(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.
Additionally, do not administer the recombinant vaccinia vaccine if the vaccinee: 

has a moderate or severe acute illness, until the illness resolves.

is undergoing topical steroid therapy for inflammatory eye diseases, or undergoing therapy 
with systemic steroids; potential immune suppression increases risk for vaccinia-related 
complications.

At this time, until a more definitive causal relationship is determined, it is recommended that 
participants 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. Participants 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. Participants 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 participants with a history of allergy to eggs or 

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egg products. Do not administer the recombinant vaccinia vaccine to participants 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. 
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. 

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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
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:
1. 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.
2. 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.
3. 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, 
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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. 
4. 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. 
5. 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. 

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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. 
6. 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, 
lymphoproliferative 
disease, 
immunosuppressive 
treatment, 
or 
HIV 
infection. 
Management should include aggressive VIG therapy.
7. 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.   
8. 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.
9. 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, 

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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 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.
Treatment of Vaccinia Vaccination Complications
Vaccinia Immune Globulin (VIG):  First-line treatment of some of the complications of vaccinia 
caused by dissemination of vaccinia virus (severe cases of inadvertent inoculation involving 
extensive lesions or if comorbid conditions exist, severe cases of generalized vaccinia in patients 
that are systemically ill and whose condition might be toxic or who have serious underlying 
immunosuppressive illnesses, eczema vaccinatum, and progressive vaccinia) is with VIG. VIG is 
contraindicated, however, for the treatment of isolated vaccinial keratitis. VIG is a sterile solution 
of the immunoglobulin fraction of pooled plasma from individuals inoculated with vaccinia 
vaccine. VIG is only available through the CDC’s Strategic National Pharmaceutical Stockpile by 
contacting the CDC’s Clinician Information Line at 1-877-554-4625 or the Director's Emergency 
Operations Center (DEOC) at 770-488-7100. Upon receipt of a call from a patient or upon direct 

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observation of a patient or contact who manifests signs and symptoms of any of the above 
conditions, the investigator should place a call to the CDC as soon as possible: 1) to initiate review 
of the clinical case, 2) to seek consultation on the appropriateness of VIG therapy, 3) to determine 
the appropriate VIG dose and dosing method for administration, if VIG therapy is required, and 4) 
to determine how to access and have the appropriate doses of VIG delivered. Early institution of 
VIG therapy is advised following recognition of clinical symptoms compatible with some vaccinia 
complications (eczema vaccinatum, severe generalized vaccinia, progressive vaccinia, and some 
cases of inadvertent inoculation). The effectiveness of VIG therapy appears to be time dependent. 
VIG has not proven to be of benefit in the treatment of post-vaccinial encephalitis, and is 
contraindicated for treatment of isolated vaccinial keratitis due to the increased risk of corneal 
scarring. A new IV formulation of VIG that has a lower level of aggregated protein allowing it to 
be used by either the IM or IV route is available through the CDC. This formulation will most 
likely be preferred for administration and the CDC will instruct investigators regarding appropriate 
dosing and method of administration based on the formulation and availability. There is no 
guarantee that VIG will successfully treat complications. At present, there are no other anti-viral 
therapies of proven benefit for the treatment of vaccinia-related complications.
14.3 CV301
14.3.1 Source 
CV301 (MVA-BN-CV301 and FPV-CV-301) will be supplied by the manufacturer, Bavarian 
Nordic.
14.3.2 Toxicity 
Updated clinical data has been presented in Table 4.
Table 4. Suspected Adverse Drug Reactions Reported by ≥1% of Subjects in the 
Completed MVA-BN Clinical Trialsa (N=8992b)

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A phase I study was just completed at the NCI with no dose limiting toxicities (unpublished). As 
expected, CV-301 was well tolerated in all 12 patients with no DLT noted. The only attributable 
toxicities were grade 1 and 2, and primarily injections site reactions and flu-like symptoms, with 
other rare toxicities such as headache and nausea (also grade 2 or less) also reported. There were 
no grade 3 or greater toxicities reported in the study. 
Initial safety data show that the most frequently occurring treatment-related AEs were temporary 
and self-limiting, grade 1 or 2 in severity and included injection site reactions (injection site 
erythema, pruritus, pain, induration and swelling) and general symptoms including fever/chills, 
flu-like symptoms, headache, fatigue/weakness, nausea/vomiting, myalgia and arthralgia. There 
was no occurrence of any of the pre-specified adverse events of special interest (immune-related 
events and cardiac events). There were no related SAEs. There were no dose limiting toxicities.
MVA-BN-CV301 (Prime Vaccine)
The most common side effects associated with MVA-BN and other MVA-BN-derived vaccines 
include mild to moderate flu-like symptoms such as: fever, chills, muscle or joint ache, and 
tiredness (fatigue). In addition, some localized reactions at the site where the vaccine is injected 
under skin (subcutaneously). These injection site reactions may include any or all of the following: 
swelling, localized pain, hardness of a small area of skin around the injection site induration), and 
redness. 

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FPV-CV301 (Boost Vaccine)
The most common side effects from the use of fowlpox vaccines are mild and may include the 
following: fever, tiredness (fatigue), low red blood cell count (anemia), low white blood cell count 
(leucopenia). 
In addition, some localized reactions at the site where the vaccine is injected under skin 
(subcutaneously). These injection site reactions may include any or all of the following: swelling, 
localized pain, hardness of a small area of skin around the injection site induration), and redness. 
14.3.3 Formulation and preparation 
MVA-BN-CV301 and FPV-CV301 encode the human MUC-1 and the human CEA gene in 
combination with human TRICOM. No marker gene is present in both recombinant viruses.
MVA-BN-CV301 is a liquid-frozen, highly attenuated, live recombinant virus based on the viral 
vector MVA-BN. It is administered as s.c. application. Packaging and vials will be labeled 
according to the respective product specifications. 
One MVA-BN-CV301 vaccine vial has a nominal titer of 4 x 108 infectious units (Inf.U) in 0.5 
mL of the drug product.
FPV-CV301 is a liquid-frozen, highly attenuated, live recombinant virus. It is administered as s.c. 
application. The packages and vials will be labeled according to the respective product 
specifications. 
One FPV-CV301 vaccine vial has a nominal virus titer of 1 x 109 Inf.U in 0.5 mL of the drug 
product.
MVA-BN-CV301 and FPV-CV301 are each supplied in 2 mL type I borosilicate glass vials closed 
with sterile bromobutyl rubber stoppers, crimped with aluminum caps and covered with 
polypropylene closures.
14.3.4 Stability and Storage 
Supplies of both the MVA-BN-CV301 and the FPV-CV301 vaccines will be shipped temperature 
controlled and monitored to the clinical trial site. Once at the site, the package should be handed 
over to personnel in charge of vaccine preparation (e.g., the pharmacist or representative). Site 
personnel are responsible for proper storage of vaccine upon receipt.
Both the MVA-BN-CV301 and the FPV-CV301 vaccines must be shipped to site and stored at a 
temperature of -80℃. A vial must not be re-frozen once it has been thawed. 
14.3.5 Administration procedures
Each dose of MVA-BN-CV301 consists of 4 injections administered subcutaneously (one in each 
arm and one in each leg). 
Each dose of FPV-CV301 consists of 1 injection administered subcutaneously (preferably in the 
non-dominant arm).
14.4 MSB0011359C (M7824, BINTRAFUSP ALFA)
Effective with amendment v7/29/2021, MSB0011359C will no longer be given as part of the 
treatment combination for the Biochemical Recurrence cohort (Arms 2 and 3).

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14.4.1 Source 
MSB0011359C (M7824) will be supplied by the manufacturer, EMD Serono.
14.4.2 Toxicity 
Updated clinical data has been presented in Table 5 and Table 6. 
Table 5. Adverse Drug Reactions by Preferred Term in the Pooled Safety Analysis Set
Pooled Safety Analysis Set
(N=765)
System Organ Class 
Preferred Term
All Grades
n (%)
Grade ≥ 3
n (%)
Blood and lymphatic system disorders
Anaemia
222 (29.0)
132 (17.3)
Blood loss anaemia
2 (0.3)
1 (0.1)
Disseminated intravascular coagulation
2 (0.3)
2 (0.3)
Haemolytic anaemia
1 (0.1)
1 (0.1)
Increased tendency to bruise
1 (0.1)
0
Microcytic anaemia
1 (0.1)
0
Normocytic anaemia
1 (0.1)
1 (0.1)
Cardiac disorders
Myocarditis *
1 (0.1)
1 (0.1)
Ear and labyrinth disorders 
Ear haemorrhage
2 (0.3)
0
Endocrine disorders
Adrenal insufficiency *
14 (1.8)
6 (0.8)
Autoimmune thyroiditis *
1 (0.1)
0
Basedow's disease *
1 (0.1)
0
Hyperthyroidism *
4 (0.5)
0
Hypophysitis *
3 (0.4)
2 (0.3)
Hypopituitarism *
2 (0.3)
1 (0.1)
Hypothyroidism *
33 (4.3)
2 (0.3)
Lymphocytic hypophysitis *
1 (0.1)
0
Secondary adrenocortical insufficiency *
1 (0.1)
0
Thyroiditis *
Eye disorders
3 (0.4)
0
Conjunctival haemorrhage
6 (0.8)
0
Retinal haemorrhage
2 (0.3)
0
Vitreous haemorrhage
1 (0.1)
1 (0.1)
Gastrointestinal disorders
Abdominal pain
111 (14.5)
15 (2.0)
Anal haemorrhage
1 (0.1)
0
Autoimmune colitis *
2 (0.3)
2 (0.3)
Colitis *
5 (0.7)
3 (0.4)
Constipation
137 (17.9)
3 (0.4)
Diarrhoea
105 (13.7)
5 (0.7)

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Pooled Safety Analysis Set
(N=765)
System Organ Class 
Preferred Term
All Grades
n (%)
Grade ≥ 3
n (%)
Diarrhoea *
8 (1.0)
5 (0.7)
Diverticulum intestinal haemorrhagic
1 (0.1)
1 (0.1)
Duodenal ulcer haemorrhage
1 (0.1)
1 (0.1)
Enterocolitis *
1 (0.1)
0
Gastric haemorrhage
2 (0.3)
1 (0.1)
Gastritis haemorrhagic
1 (0.1)
1 (0.1)
Gastroduodenal haemorrhage
1 (0.1)
1 (0.1)
Gastrointestinal haemorrhage
14 (1.8)
8 (1.0)
Gastrointestinal vascular malformation haemorrhagic
1 (0.1)
1 (0.1)
Gingival bleeding
38 (5.0)
0
Haematemesis
6 (0.8)
1 (0.1)
Haematochezia
5 (0.7)
0
Haemorrhagic ascites
1 (0.1)
0
Haemorrhoidal haemorrhage
8 (1.0)
2 (0.3)
Intra-abdominal haematoma
1 (0.1)
1 (0.1)
Intra-abdominal haemorrhage
3 (0.4)
3 (0.4)
Lower gastrointestinal haemorrhage
4 (0.5)
1 (0.1)
Melaena
12 (1.6)
2 (0.3)
Mouth haemorrhage
9 (1.2)
0
Nausea
137 (17.9)
12 (1.6)
Oesophageal haemorrhage
1 (0.1)
1 (0.1)
Oesophageal varices haemorrhage
2 (0.3)
2 (0.3)
Rectal haemorrhage
5 (0.7)
0
Small intestinal haemorrhage
1 (0.1)
0
Upper gastrointestinal haemorrhage
14 (1.8)
8 (1.0)
Vomiting
100 (13.1)
11 (1.4)
General disorders and administration site conditions
Asthenia
126 (16.5)
15 (2.0)
Chills
3 (0.4)
0
Fatigue
160 (20.9)
24 (3.1)
Mucosal haemorrhage
2 (0.3)
0
Oedema peripheral
79 (10.3)
2 (0.3)
Pyrexia
141 (18.4)
7 (0.9)
Hepatobiliary disorders
Hepatic function abnormal *
1 (0.1)
1 (0.1)
Hepatic haemorrhage
1 (0.1)
1 (0.1)
Hepatitis *
2 (0.3)
1 (0.1)
Immune system disorders
Drug hypersensitivity
1 (0.1)
0
Injury, poisoning and procedural complications
1 (0.1)
0
Contusion
9 (1.2)
1 (0.1)
Extradural haematoma
1 (0.1)
1 (0.1)
Infusion related reaction
26 (3.4)
1 (0.1)
Subcutaneous haematoma
1 (0.1)
0
Subdural haematoma
1 (0.1)
1 (0.1)
Investigations
Alanine aminotransferase increased *
6 (0.8)
4 (0.5)
Aspartate aminotransferase increased
87 (11.4)
19 (2.5)
Aspartate aminotransferase increased *
6 (0.8)
5 (0.7)

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Pooled Safety Analysis Set
(N=765)
System Organ Class 
Preferred Term
All Grades
n (%)
Grade ≥ 3
n (%)
Blood creatine phosphokinase increased *
2 (0.3)
1 (0.1)
Blood thyroid stimulating hormone increased *
2 (0.3)
0
Haemoglobin decreased
4 (0.5)
3 (0.4)
Transaminases increased *
1 (0.1)
0
Metabolism and nutrition disorders
Decreased appetite
189 (24.7)
18 (2.4)
Latent autoimmune diabetes in adults *
1 (0.1)
1 (0.1)
Type 1 diabetes mellitus *
1 (0.1)
0
Musculoskeletal and connective tissue disorders
Back pain
1 (0.1)
0
Myositis *
2 (0.3)
2 (0.3)
Neoplasms benign, malignant and unspecified (incl cysts 
and polyps)
Basal cell carcinoma
6 (0.8)
1 (0.1)
Bowen's disease
2 (0.3)
2 (0.3)
Intracranial tumour haemorrhage
1 (0.1)
1 (0.1)
Keratoacanthoma
57 (7.5)
5 (0.7)
Lip squamous cell carcinoma
4 (0.5)
2 (0.3)
Skin neoplasm bleeding
1 (0.1)
0
Squamous cell carcinoma of skin
27 (3.5)
11 (1.4)
Tumour haemorrhage
13 (1.7)
10 (1.3)
Nervous system disorders
Cerebellar haemorrhage
1 (0.1)
1 (0.1)
Cerebral haemorrhage
1 (0.1)
1 (0.1)
Haemorrhage intracranial
3 (0.4)
3 (0.4)
Haemorrhagic transformation stroke
1 (0.1)
0
Headache
86 (11.2)
5 (0.7)
Immune-mediated encephalitis *
1 (0.1)
1 (0.1)
Renal and urinary disorders
Acute kidney injury *
3 (0.4)
3 (0.4)
Haematuria
22 (2.9)
3 (0.4)
Nephritis *
1 (0.1)
1 (0.1)
Tubulointerstitial nephritis *
1 (0.1)
1 (0.1)
Reproductive system and breast disorders
Breast haemorrhage
1 (0.1)
0
Vaginal haemorrhage
4 (0.5)
0
Respiratory, thoracic and mediastinal disorders
Bronchial haemorrhage
1 (0.1)
0
Cough
87 (11.4)
0
Dyspnoea
134 (17.5)
31 (4.1)
Epistaxis
81 (10.6)
3 (0.4)
Haemoptysis
41 (5.4)
4 (0.5)
Haemothorax
1 (0.1)
1 (0.1)
Interstitial lung disease *
4 (0.5)
2 (0.3)
Laryngeal haemorrhage
1 (0.1)
0

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Pooled Safety Analysis Set
(N=765)
System Organ Class 
Preferred Term
All Grades
n (%)
Grade ≥ 3
n (%)
Pharyngeal haemorrhage
1 (0.1)
0
Pneumonitis *
8 (1.0)
3 (0.4)
Pulmonary haemorrhage
4 (0.5)
2 (0.3)
Respiratory tract haemorrhage
1 (0.1)
0
Skin and subcutaneous tissue disorders 
Actinic keratosis
12 (1.6)
0
Blood blister
3 (0.4)
0
Dermatitis acneiform *
14 (1.8)
2 (0.3)
Drug eruption *
1 (0.1)
0
Ecchymosis
1 (0.1)
0
Erythema *
3 (0.4)
0
Hyperkeratosis
12 (1.6)
2 (0.3)
Lichen planus *
1 (0.1)
0
Pemphigoid *
3 (0.4)
3 (0.4)
Petechiae
2 (0.3)
0
Pruritus
143 (18.7)
2 (0.3)
Pruritus *
38 (5.0)
3 (0.4)
Purpura
5 (0.7)
0
Rash *
42 (5.5)
11 (1.4)
Rash erythematous *
4 (0.5)
0
Rash macular *
8 (1.0)
2 (0.3)
Rash maculo-papular *
44 (5.8)
16 (2.1)
Rash papular *
2 (0.3)
1 (0.1)
Rash pruritic *
9 (1.2)
1 (0.1)
Skin haemorrhage
1 (0.1)
0
Toxic skin eruption *
1 (0.1)
1 (0.1)
Vascular disorders 
Flushing
2 (0.3)
0
Haematoma
6 (0.8)
0
Hypotension
5 (0.7)
0
Shock haemorrhagic
1 (0.1)
1 (0.1)
Source: ADSL 23DEC2020 8:38, ADAE 23DEC2020 9:27,OutputID:T-38-aefrq.
* Immune-related adverse reaction as identified via customized MedDRA PT query and assessed based 
on a detailed medical review using predefined criteria.
Table 6. Serious Adverse Drug Reactions by Preferred Term (reported for ≥ 2 
Participants per PT) Considered Expected for the Purpose of Safety Reporting to 
Regulatory Authorities in the European Union
Pooled Safety Analysis Set 
(N=765)
System Organ Class 
Preferred Term
Treatment-related SARs
n (%)
Blood and lymphatic system disorders
Anaemia
6 (0.8)
Endocrine disorders

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Pooled Safety Analysis Set 
(N=765)
System Organ Class 
Preferred Term
Treatment-related SARs
n (%)
Adrenal insufficiency 
Hypophysitis
7 (0.9)
2 (0.3)
Gastrointestinal disorders
Colitis
Autoimmune colitis
Diarrhoea
4 (0.5)
2 (0.3)
2 (0.3)
Musculoskeletal and connective tissue disorders
Myositis
2 (0.3)
Neoplasms benign, malignant and unspecified (incl cysts and polyps)
Squamous cell carcinoma of skin
Keratoacanthoma
Lip squamous cell carcinoma
Basal cell carcinoma
Bowen's disease
19 (2.5)
10 (1.3)
3 (0.4)
2 (0.3)
2 (0.3)
Renal and urinary disorders
Acute kidney injury
2 (0.3)
Respiratory, thoracic and mediastinal disorders
Pneumonitis
Interstitial lung disease
5 (0.7)
4 (0.5)
Skin and subcutaneous tissue disorders
Rash maculo-papular
Rash
3 (0.4)
2 (0.3)
Source: ADSL 23DEC2020 8:38, ADAE 23DEC2020 9:27,OutputID:T-39-aefrq.
SAR = serious adverse reactions.
The important potential risks include hypersensitivity, irAEs/autoimmune disorders, anemia, rash 
with hyperkeratosis/keratoacanthomas/SCC of the skin, embryo-fetal toxicity, and impaired 
wound healing. The important identified risks with bintrafusp alfa observed to data were overall 
manageable. Mucosal bleeding events of mild to moderate severity were observed in participants 
treated with bintrafusp alfa in ongoing studies and are a potential risk for bintrafusp alfa. Events 
may include epistaxis, hemoptysis, gingival bleeding or hematuria amongst others. In general, 
these reactions resolve without discontinuation of treatment. 
In addition, after discussion among NCI investigators on multiple protocols using M7824, multiple 
bleeding events ranging from low grade gingival bleeding and epistaxis to more serious 
hemoptysis, GI bleeding and hematuria have been observed. Some of these events can be attributed 
to bleeding events related to cancer directly and others bleeding events can be attributed to colitis 
or cystitis which is a known toxicity of anti-PD-L1 agents including M7824. However, there 
remains the possibility that M7824 may increase the overall risk of bleeding in ways that may not 
be directly related to direct tumor bleeding or inflammatory bleeding events described with 
checkpoint inhibitors like M7824. It is hypothesized that this possible increased bleeding risk may 
be due to TGF beta inhibition which has an effect on angiogenesis; bleeding has also been observed 
in participants receiving M7824 and may be drug-related (e.g., gum bleeding, nose bleeds, 
coughing up blood, blood in their urine, or blood in the stool). Accordingly, participants will be 
notified of the same possible risk in the informed consent document for this study.

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Embryofetal toxicities are known risk of the PD 1/PD L1 targeting class and are considered 
important potential risks for MSB0011359C. Animal models link the PD 1/PD L1 signaling 
pathway with maintenance of pregnancy through induction of maternal immune tolerance to fetal 
tissue. Embryofetal toxicity is an important potential risk of MSB0011359C. An appropriate 
contraception warning is provided as part of the inclusion criteria. Pregnant and breastfeeding 
women are not allowed in the MSB0011359C study, and adequate contraceptive measures are 
recommended during the study to minimize or eliminate the potential risk to the developing fetus.
14.4.3 Formulation and preparation 
MSB0011359C is supplied as a liquid formulation packaged at a 10 mg/mL concentration in 
USP/Ph Eur Type I 50R vials that are filled with drug product solution to allow an extractable 
volume of 60 mL (600mg/mL). The liquid formulation must be diluted with 0.9% sodium chloride 
for injection solution to a total volume of 250 mL. 
14.4.4 Stability and Storage 
M7824 drug product must be stored at 2°C to 8°C until use. The storage condition is based on data 
from ongoing long term stability studies with M7824.
M7824 drug product stored at room (23°C to 27°C) or higher temperatures for extended periods 
of time might be subject to degradation. M7824 must not be frozen. Rough shaking of the 
reconstituted solution must be avoided.
The chemical and physical in-use stability for the infusion solution of M7824 in 0.9% saline 
solution has been demonstrated for a total of 72 hours at room temperature. However, from a 
microbiological point of view, the diluted solution should be used immediately and is not intended 
to be stored unless dilution has taken place in controlled and validated aseptic conditions. If not 
used immediately, in-use storage times and conditions prior to administration are the responsibility 
of the user.
No other drugs should be added to the infusion containers containing M7824.
14.4.5 Administration procedures
M7824 is administered as a 1-hour intravenous (iv) infusion once every 2 weeks. Dose, frequency, 
and administration of M7824 must be in accordance with the respective clinical study protocol.

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18.
Tsang, K.Y., et al., Analyses of recombinant vaccinia and fowlpox vaccine vectors 
expressing transgenes for two human tumor antigens and three human costimulatory 
molecules. Clin Cancer Res, 2005. 11(4): p. 1597-607.

Abbreviated Title: Combination Immunotherapy 
Version Date:  05/10/2023
89
19.
Mohebtash, M., et al., A pilot study of MUC-1/CEA/TRICOM poxviral-based vaccine in 
patients with metastatic breast and ovarian cancer. Clin Cancer Res, 2011. 17(22): p. 
7164-73.
20.
Morse, M.A., et al., A randomized phase II study of immunization with dendritic cells 
modified with poxvectors encoding CEA and MUC1 compared with the same poxvectors 
plus GM-CSF for resected metastatic colorectal cancer. Ann Surg, 2013. 258(6): p. 879-
86.
21.
Hotchkiss, R.S. and L.L. Moldawer, Parallels between cancer and infectious disease. N 
Engl J Med, 2014. 371(4): p. 380-3.
22.
Lebrun, J.J., The Dual Role of TGFbeta in Human Cancer: From Tumor Suppression to 
Cancer Metastasis. ISRN Mol Biol, 2012. 2012: p. 381428.
23.
Wrzesinski, S.H., Y.Y. Wan, and R.A. Flavell, Transforming growth factor-beta and the 
immune response: implications for anticancer therapy. Clin Cancer Res, 2007. 13(18 Pt 
1): p. 5262-70.
24.
Hao, N.B., et al., Macrophages in tumor microenvironments and the progression of 
tumors. Clin Dev Immunol, 2012. 2012: p. 948098.
25.
Kattan, M.W., et al., The addition of interleukin-6 soluble receptor and transforming 
growth factor beta1 improves a preoperative nomogram for predicting biochemical 
progression in patients with clinically localized prostate cancer. J Clin Oncol, 2003. 
21(19): p. 3573-9.
26.
Schroten, C., et al., The additional value of TGFbeta1 and IL-7 to predict the course of 
prostate cancer progression. Cancer Immunol Immunother, 2012. 61(6): p. 905-10.
27.
Souza-Fonseca-Guimaraes, F. and M.J. Smyth, Myeloid TGF-beta responsiveness 
promotes metastases. Cancer Discov, 2013. 3(8): p. 846-8.
28.
Suzuki, E., et al., Soluble type II transforming growth factor-beta receptor inhibits 
established murine malignant mesothelioma tumor growth by augmenting host antitumor 
immunity. Clin Cancer Res, 2004. 10(17): p. 5907-18.
29.
Donkor, M.K., A. Sarkar, and M.O. Li, Tgf-beta1 produced by activated CD4(+) T Cells 
Antagonizes T Cell Surveillance of Tumor Development. Oncoimmunology, 2012. 1(2): 
p. 162-171.
30.
Topalian, S.L., C.G. Drake, and D.M. Pardoll, Targeting the PD-1/B7-H1(PD-L1) 
pathway to activate anti-tumor immunity. Curr Opin Immunol, 2012. 24(2): p. 207-12.
31.
Song, S., et al., Dendritic cells with an increased PD-L1 by TGF-beta induce T cell 
anergy for the cytotoxicity of hepatocellular carcinoma cells. Int Immunopharmacol, 
2014. 20(1): p. 117-23.
32.
Gibney, G.T., et al., Safety, correlative markers, and clinical results of adjuvant 
nivolumab in combination with vaccine in resected high-risk metastatic melanoma. Clin 
Cancer Res, 2015. 21(4): p. 712-20.
33.
Gulley, J.L., Therapeutic vaccines: the ultimate personalized therapy? Hum Vaccin 
Immunother, 2013. 9(1): p. 219-21.
34.
Boehm, A.L., et al., Concurrent vaccination with two distinct vaccine platforms targeting 
the same antigen generates phenotypically and functionally distinct T-cell populations. 
Cancer Immunol Immunother, 2010. 59(3): p. 397-408.
35.
Beer, T.M., et al., Randomized, Double-Blind, Phase III Trial of Ipilimumab Versus 
Placebo in Asymptomatic or Minimally Symptomatic Patients With Metastatic 

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90
Chemotherapy-Naive Castration-Resistant Prostate Cancer. J Clin Oncol, 2017. 35(1): p. 
40-47.
36.
Kwon, E.D., et al., Ipilimumab versus placebo after radiotherapy in patients with 
metastatic castration-resistant prostate cancer that had progressed after docetaxel 
chemotherapy (CA184-043): a multicentre, randomised, double-blind, phase 3 trial. 
Lancet Oncol, 2014. 15(7): p. 700-12.
37.
Graff, J.N., et al., Early evidence of anti-PD-1 activity in enzalutamide-resistant prostate 
cancer. Oncotarget, 2016. 7(33): p. 52810-52817.
38.
Gajewski, T.F. and T. Schumacher, Cancer immunotherapy. Curr Opin Immunol, 2013. 
25(2): p. 259-60.
39.
Larkin, J., et al., Combined Nivolumab and Ipilimumab or Monotherapy in Untreated 
Melanoma. N Engl J Med, 2015. 373(1): p. 23-34.
40.
Fu, J., et al., Preclinical evidence that PD1 blockade cooperates with cancer vaccine 
TEGVAX to elicit regression of established tumors. Cancer Res, 2014. 74(15): p. 4042-
52.
41.
Gulley, J.L., et al., Role of Antigen Spread and Distinctive Characteristics of 
Immunotherapy in Cancer Treatment. J Natl Cancer Inst, 2017. 109(4).
42.
Vergati, M., et al., Analysis of circulating regulatory T cells in patients with metastatic 
prostate cancer pre- versus post-vaccination. Cancer Immunol Immunother, 2011. 60(2): 
p. 197-206.
43.
Farsaci, B., et al., Identification by digital immunohistochemistry of intratumoral changes 
of immune infiltrates after vaccine in the absence of modifications of PBMC immune cell 
subsets. Int J Cancer, 2014. 135(4): p. 862-70.
44.
Roth, A., et al., Assessing anxiety in men with prostate cancer: further data on the 
reliability and validity of the Memorial Anxiety Scale for Prostate Cancer (MAX-PC). 
Psychosomatics, 2006. 47(4): p. 340-7.
45.
Roth, A.J., et al., The memorial anxiety scale for prostate cancer: validation of a new 
scale to measure anxiety in men with with prostate cancer. Cancer, 2003. 97(11): p. 
2910-8.
46.
Victorson, D.E., et al., Efficient assessment of the most important symptoms in advanced 
prostate cancer: the NCCN/FACT-P Symptom Index. Psychooncology, 2011. 20(9): p. 
977-83.

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16 APPENDIX A: PERFORMANCE STATUS CRITERIA
ECOG Performance Status Scale
Karnofsky Performance Scale
Grade
Descriptions
Percent
Descriptions
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 light or sedentary 
work (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 
> 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 > 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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17 APPENDIX B: VACCINIA-PSA(L155)/TRICOM 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 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 blisters on day 5 to 6 and then 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.
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.

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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 unbandaged 
site because live virus could be washed onto other areas on 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 7-14 days after vaccination) there are several kinds of people with 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). 

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18 APPENDIX C: ON STUDY AND FOLLOW-UP EVALUATIONS
18.1 STUDY CALENDAR: BIOCHEMICAL RECURRENCE COHORT
Screening 
Baseline
Surveillance for 4 months20
Vaccines for 7 months
End of 
treatment1
Follo
w-
up14
M -4   M -3
M-2
M-1
C1
D1
C
1
D
15
C2
D1
C3
D1
C4
D1
C5
D1
C6
D1
C7
D1
Informed
Consent / 
Registration
X
NIH Advance 
Directives Form13
X
TREATMENT
Prostvac-V2
X
Prostvac-F3
X
X
X
X
X
X
X
MVA-BN-CV3014
X
X
FPV-CV3015
X
X
X
X
X
X
EVALUATION
History and PE, 
Weight
X8
X9
X
X
X
X
X
X
X
X
X
X
X
X
ECOG Performance 
Score
X7
X9
Pathologic 
confirmation of dx
X6
Laboratory Testing
CBC with 
differential
X8
X9
X
X
X
X
X
X
X
X
X
X
Acute Care Panel
X8
Hepatic Panel
X8

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Screening 
Baseline
Surveillance for 4 months20
Vaccines for 7 months
End of 
treatment1
Follo
w-
up14
M -4   M -3
M-2
M-1
C1
D1
C
1
D
15
C2
D1
C3
D1
C4
D1
C5
D1
C6
D1
C7
D1
Serum chemistries 
(Na+, K+, Cl-, CO2, 
glucose, BUN, 
creatinine, albumin,
calcium, 
magnesium, 
phosphorus, alkaline 
phosphatase, ALT, 
AST, total bilirubin, 
LDH, CK, uric acid, 
total protein)
X9
X
X
X
X
X
X
X
X
X
X
Serum amylase and 
lipase
X9
X
X
X
X
X
X
X
X
X
X
Serum thyroid 
stimulating hormone 
(TSH)
X9
X
X
X
X
X
X
X
X
X
X
Urinalysis
X9
X
Serum PSA and 
Testosterone
X8
X9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Hep B/Hep C, HIV
X7
Lymphocyte 
Phenotyping 
(CD4/CD8)
X9
X
X
X
X
X
X
X
X
X
X
PAP (prostatic acid 
phosphatase)
X9
X
X
X
X
X
X
X
X
X
X
ECG
X10
Tc 99 scintigraphy
X7
X21
X
X11
CT-C/A/P
X7
X21
X
X11
18F-DCFPyL PET 
scan
X19
X19

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Screening 
Baseline
Surveillance for 4 months20
Vaccines for 7 months
End of 
treatment1
Follo
w-
up14
M -4   M -3
M-2
M-1
C1
D1
C
1
D
15
C2
D1
C3
D1
C4
D1
C5
D1
C6
D1
C7
D1
NaF PET scan
X15
X15
Research Bloods
X16
X16
X
X12
X12
Research stool 
sample collection 
(optional) 18
X
X
X
Adverse Events
X
 
X
X
 
X
X
Concomitant 
Medications
X
 
 
X
X
FACT-P survey
X17
X17
X17
X17
MSK Anxiety 
Survey
X17
X17
X17
X17
1 cycle = 28 days; C=Cycle; D=Day; M = month (28 days)
Participant visits may be delayed for up to 2 weeks for logistical reasons during the surveillance period. While participant is receiving 
immunotherapy, visits and treatment may be -/+3 days in the first cycle around the schedule visit. Participants being evaluated after cycle 1 may 
have their follow-up visits adjusted by +/-7 days for logistical purposes. Laboratory tests specified for each cycle may be performed up to 5 days 
prior to the start of each cycle for logistical purposes. For participants who do not receive treatment during this window, the dose will be 
considered missed and the next visit will occur at the next scheduled time. 
Footnotes:
1 End of treatment visit will occur 28 days (-/+14 days) following the last dose of immunotherapy. Willing participants will continue to be followed 
after cycle 7 until progression or ADT is clinically indicated. 
2 Prostvac-V 2 x 108 infectious units/ 0.5 mL by subcutaneous injection.
3 Prostvac-F 1 x 109 infectious units/0.5 mL by subcutaneous injection.
4 MVA-BN-CV301: one dose is four sites of subcutaneous injection each consisting of 4 x 108 infectious units/0.5 mL.
5 FPV-CV301 1 x 109 infectious units/0.5 mL by subcutaneous injection.
6 Pathologic confirmation will be obtained any time prior to enrollment.
7 Screening assessments obtained within 8 weeks prior to enrollment.
8 Screening assessments obtained within 16 days prior to start of enrollment.

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9 Baseline assessments obtained within 16 days prior to treatment (these tests will not need to be repeated if they were done at screening within the 
appropriate timeframe).
10 Baseline ECG will be obtained within 8 weeks prior to enrollment
11 Follow-up Imaging will be done prior to vaccine therapy (cycle 1), and at the end of study visit. During the follow-up period, imaging will be done 
every 6 months for 1 year then as clinically indicated. A window of +/- 21 days for imaging is allowed. Restaging scans may be completed earlier if 
clinically indicated. 
12 Optional
13 As indicated in section 12.3, all subjects will be offered the opportunity to complete an NIH advance directives form. This should be done preferably 
at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is strongly 
recommended but is not required.
14 See Section  3.4 for further details on follow up visits.
15 For participants in the BCR cohort, two NaF PET Scans will be done when logistically feasible. These scans are for research purposes only. These 
scans will not be used to evaluate for clinical progression of disease.
16 Can be done any time prior to first treatment.
17 Surveys are optional at all time points and can be done as noted on study calendar, and every 3 months thereafter while participants are on-study. If 
surveys are not done, reason will be documented.
18 Participants will be asked to give stool samples during the surveillance period, after 3-4 months on treatment and after completion of  7 months of 
treatment  (Section 5.2.3). Note: These are optional.
19 When logistically feasible, PSMA PET scans should be done in the first two months during surveillance (or within 4 months of treatment initiation if 
surveillance period is shorter per Amendment v05/10/2023 and within 8 weeks after completing treatment. If PSMA PET is obtained within 8 weeks 
of starting surveillance, it will not need to be repeated during the surveillance period. 
20 With Amendment v05/10/2023, participants that have at least 3 PSA measurements in the surveillance period, with at least one of them sufficient to 
provide the doubling time from baseline, will not undergo any further PSA measurements in the surveillance period.
21 Participants starting treatment per Amendment v05/10/2023 with a surveillance period of 8 weeks or shorter do not need to repeat CT and Tc99 imaging 
on C1D1 
 

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18.2 STUDY CALENDAR: CRPC LEAD-IN COHORT 
This cohort is closed as of December 2018.
Screening
Baseline
Vaccines for 4 months
Vaccines plus MSB001359C for 
3 months
End of 
treatment1
Safety 
Visit15
C1
D1
C1
D15
C2
D1
C2
D15
C3
D1
C3
D15
C4
D1
C5
D1
C6
D1
C7
D1
C8
D1
C9+
Informed
Consent / Registration
X
NIH Advance 
Directives Form14
X
TREATMENT
Prostvac-V2
X
Prostvac-F3
X
X
 
X
 
X
X
X
X
X
X
MVA-BN-CV3014
X
X
FPV-CV3015
X
 
X
 
X
X
X
X
X
X
MSB001359C6
X
X
X
X
X
X
 
 
EVALUATION
History and PE, 
Weight
X9
X10
X
X
X
X
X
X
X
X
X
X
X
X
X
X
ECOG Performance 
Score
X8
X10
Pathologic 
confirmation of dx
X7
Laboratory Testing
CBC with differential
X9
X10
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Serum chemistries 
(Na+, K+, Cl-, CO2, 
glucose, BUN, 
creatinine, albumin,
calcium, magnesium, 
phosphorus, alkaline 
phosphatase, ALT, 
AST, total bilirubin, 
X10
X
X
X
X
X
X
X
X
X
X
X
X
X
X

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100
1 cycle = 28 days; C=Cycle; D=Day; M = month (28 days)
Participant visits may be delayed for up to 2 weeks for logistical reasons during the surveillance period. While participant is receiving 
immunotherapy, visits and treatment may be -/+3 days in the first cycle around the schedule visit. Participants being evaluate after cycle 1 may 
have their follow-up visits adjusted by +/-7 days for logistical purposes. Laboratory tests specified for each cycle may be performed up to 5 days 
prior to the start of each cycle for logistical purposes. For participants who do not receive treatment during this window, the dose will be 
considered missed and the next visit will occur at the next scheduled time. 
Footnotes:
1
End of treatment visit will occur 28 days (+/- 7 days) after last dose of study treatment when logistically feasible and participants are willing. 
LDH, CK, uric acid, 
total protein)
Serum amylase and 
lipase
X10
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Serum thyroid 
stimulating hormone 
(TSH)
X10
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Urinalysis
X10
X
Serum PSA and 
Testosterone
X9
X10
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Hep B/HepC, HIV
X8
Lymphocyte 
Phenotyping 
(CD4/CD8)
X10
X
X
X
X
X
X
X
X
X
X
X
X
X
PAP (prostatic acid 
phosphatase)
X10
X
X
X
X
X
X
X
X
X
X
X
X
X
X
ECG
X11
Tc 99 scintigraphy
X8
X
X
X
X12
X
CT-C/A/P, or MRI
X8
X
X
X
X12
Research Bloods
X16
X
X
X
X
X
X13
X13
Adverse Events
X
X
X
Concomitant 
Medications
X
X
X

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2 Prostvac-V 2 x 108 infectious units/ 0.5 mL by subcutaneous injection.
3 Prostvac-F 1 x 109 infectious units/0.5 mL by subcutaneous injection.
4 MVA-BN-CV301: one dose is four sites of subcutaneous injection each consisting of 4 x 108 infectious units/0.5 mL.
5 FPV-CV301 1 x 109 infectious units/0.5 mL by subcutaneous injection.
6 MSB0011359C (anti-PDL1 TGFβ Trap) 1200mg by intravenous infusion over 60 minutes.
7 Pathologic confirmation will be obtained any time prior to enrollment.
8 Screening assessments obtained within 8 weeks prior to enrollment.
9 Screening assessments obtained within 16 days prior to start of enrollment.
10 Baseline assessments obtained within 16 days prior to treatment (these tests will not need to be repeated if they were done at screening within the 
appropriate timeframe).
11 Baseline ECG will be obtained within 8 weeks prior to enrollment.
12 Restaging will be done every 3 months after cycle 9 and at the safety visit. After the safety visit the participant will be removed from study. 
13 Optional
14 As indicated in section 12.3, all subjects will be offered the opportunity to complete an NIH advance directives form. This should be done preferably 
at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is strongly 
recommended, but is not required.
15 See Section 3.4 for further details on follow up visits.
16 Can be done any time prior to first treatment.

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19 APPENDIX D: INSTRUCTIONS FOR PRE-STUDY AND FOLLOW-UP BLOOD TESTS
Blood Studies
Blood Tube/Comments
Destination
CBC with differential
1 light lavender tube
CC Department of Laboratory Medicine 
(DLM)
Hepatic Panel, Mineral Panel, Acute Care 
Panel, LDH, CK, Uric Acid, Total Protein
1 4 mL SST
CC DLM
Anti-HIV-1/2
1 8 mL SST
CC TTV lab
Testosterone, total 
1 red top tube
CC DLM
Prostate Specific Antigen
4 mL SST
CC DLM
Lymphocyte Phenotyping, TBNK
1 light lavender tube
CC DLM
Immunology Assays
6 10 mL Na Heparin tubes
2 8 ml SST tubes
NCI-Frederick
ADL = activities of daily living; ALT = alanine aminotransferase; AST = aspartate aminotransferase; CT = computed tomography; irAE = immune-related adverse event; IV = 
intravenous; LFT = liver function test; LLN = lower limit of normal; MRI = magnetic resonance imaging; NCI-CTCAE = National Cancer Institute-Common Terminology Criteria 
for Adverse Events; T4 = free thyroxine; TSH = thyroid-stimulating hormone; ULN = upper limit of normal.

Abbreviated Title: Combination Immunotherapy 
Version Date:  05/10/2023
104
FOR BROWN TOP CONTAINER
7) Use the small shovel attached to the lid of the brown top container to fill container 
approximately 1/2 full. 
8) Close the brown top container lid and secure the tightly.
9) Dispose of any remaining feces into the toilet. 
10)Seal the lid of the plastic bowl and put the whole container in to the plastic garbage bag 
provided. 
11)Throw gloves away (if used) in the same garbage bag. 
12)Tie the garbage bag securely and throw away with normal trash. 
13)Wash Hands.
14)Put the brown top container in the small plastic biohazard bag provided.
15)Freeze the brown top container for at least 4 hours until you are ready to bring to clinic 
or ship.
16) To bring to clinic, place specimen in the cooler bag, along with the frozen ice packs 
provided. To ship, please see shipping instructions.

Abbreviated Title: Combination Immunotherapy 
Version Date:  05/10/2023
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21 APPENDIX G:  PURPLE TOP STOOL COLLECTION INSTRUCTIONS

Abbreviated Title: Combination Immunotherapy 
Version Date:  05/10/2023
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22 APPENDIX H: STOOL SPECIMEN SHIPMENT INSTRUCTIONS 
Shipping Material:

There is a white cardboard box that you will be working with. 

There is a white Styrofoam box in the white cardboard box with a piece of bubble 
wrap on top. 

Keep these for return shipping.
          

Inside the Styrofoam box is an insulated silver bag with an ice pack inside – 
place this entire bag in the freezer. 
        
                                                
Follow specific collection instructions for purple and brown top tubes 
provided in separate handouts.

Abbreviated Title: Combination Immunotherapy 
Version Date:  05/10/2023
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PURPLE TOP CONTAINER

After tightly securing the lid, write the date and time of the collection on the 
container. 

Wrap the container in the white absorbent material and place in the clear plastic 
Biohazard specimen bag.

Zip seal the specimen bag and place in the freezer for at least 4 hours prior to 
shipping.

Keep the purple top container frozen until you ship the whole box back 
Ship the box back as soon as you can. 
           
  
    
  
                              
BROWN TOP CONTAINER

After placing the stool in the brown top container, write the date and time of the 
collection on the container. 

Wrap the container in the absorbent material inside the specimen bag and place 
the container inside the specimen bag.

Seal the specimen bag according to the instructions written on the bag and place 
in the freezer for at least 4 hours prior to shipping. 

Keep the brown top container frozen until you ship the whole box back.  
Ship the box back as soon as you can. 

Abbreviated Title: Combination Immunotherapy 
Version Date:  05/10/2023
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Just prior to shipping:

Place the specimen bag containing the FROZEN brown and purple tops inside 
the insulated silver bag that has been in your freezer. 

This will go inside the Styrofoam container.

Place the Styrofoam box inside the white cardboard box.

Place the bubble wrap on top of the Styrofoam box, and tape white cardboard 
box closed.

Abbreviated Title: Combination Immunotherapy 
Version Date:  05/10/2023
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
Mail the white cardboard box back. 

The return FedEx label has already been filled out, including our FedEx account 
number for billing. 

Fill in the date only on the return FedEx label.

Drop the entire box off at a FedEx facility, or call 1-800-463-3339 to schedule a 
pick-up. 

Do not ship it on a Thursday, Friday, or before a holiday. The box cannot be 
delivered on the weekend.