Clinical trial NCT04057365
Conditions: Biliary Tract Cancer
Keywords: Biliary Tract Cancer
Sponsor: Massachusetts General Hospital
Start date: 2019-10-07; Completion date: 2022-09-25
Enrollment: 15 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This research is studying the effect of the combination of how two study drugs (Nivolumab and DKN-01) works in people with advanced biliary tract cancer.

Detailed description:
This research study is a Phase II clinical trial. Phase II clinical trials test the safety and effectiveness of an investigational drug to learn whether the drug works in treating a specific disease. "Investigational" means that the drug is being studied.

The U.S. Food and Drug Administration (FDA) has not approved DKN-01 as a treatment for any disease.

The FDA has not approved nivolumab for this specific disease but it has been approved for other cancers.

DKN-01 and nivolumab are both antibodies. An antibody is a protein that attaches to other cells to fight off infection. DKN-01 is believed to work by attaching to and inhibiting (stopping) a specific pathway in the cells that is responsible for processes such as cell growth. Nivolumab is believed to work by attaching to and inhibiting a specific protein in the cancer that controls parts of the immune system (the system in the body that fights off infections and diseases) by shutting down certain immune responses. The investigators believe that nivolumab will inhibit the protein, thus allowing the immune cells to recognize and destroy cancer cells.

Arms: arm 1: * DKN-01 will be administered intravenously on day 1 and day 15 of each 28 day cycle
* Nivolumab will be administered intravenously on day 1 and day 15 of each 28 day cycle arm 2: * DKN-01 will be administered intravenously on day 1 and day 15 of each 28 day cycle
* Nivolumab will be administered intravenously on day 1 and day 15 of each 28 day cycle
Interventions: intervention 1: Nivolumab intervention 2: DKN-01
Intervention descriptions: intervention 1: Nivolumab is believed to work by attaching to and inhibiting a specific protein in the cancer that controls parts of the immune system (the system in the body that fights off infections and diseases) by shutting down certain immune responses. The investigators believe that nivolumab will inhibit the protein, thus allowing the immune cells to recognize and destroy cancer cells. intervention 2: DKN-01 is believed to work by attaching to and inhibiting (stopping) a specific pathway in the cells that is responsible for processes such as cell growth

Protocol documents:
Prot_SAP_000.pdf: 
Page 1
NCI Protocol #:
Assigned by the NCI.  
BMS Protocol Number: CA209-76J
DF/HCC Protocol #: 19-200
TITLE:  A Single Arm Phase II Study of the Combination of DKN-01 and Nivolumab in 
Previously Treated Patients with Advanced Biliary Tract Cancer (BTC)
Coordinating Center:
Massachusetts General Hospital Cancer Center
*Principal Investigator (PI):
 Joseph W. Franses, MD., PhD
Massachusetts General Hospital Cancer Center
 jfranses@mgh.harvard.edu
Other Investigators:
Thomas Abrams, MD
Dana Farber Cancer Institute 
Thomas_Abrams@dfci.harvard.edu
Statistician:
      Hui Zheng
Massachusetts General Hospital
HZHENG1@PARTNERS.ORG
Responsible Research Nurse:
Responsible Data Manager: 
Angelo Gemma 
      Patricia Lynch, RN
 Eamala Sundaram
Massachusetts General Hospital
Massachusetts General Hospital
32 Fruit St.
125 Nashua St., suite 940
617-643-0816
Agents:  DKN-01, NSC # (if applicable), Leap Therapeutics
               Nivolumab, NSC # (if applicable), Bristol Myers Squibb
IND #: 144535 
IND Sponsor:  Joseph W. Franses, MD., PhD
Amendments: 
Amendment 1 – September 4, 2019
Amendment 2 – January 8, 2020
Amendment 3 – May 01, 2020
Amendment 4 – May 09, 2023
NCT04057365


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SCHEMA
Lead-in DLT period (3 participants):
DKN-01 Dose Level 1 and Nivolumab  IV, both 
on Day 1 and 15 of each 28 day cycle
If 1 DLT, recruit 3 more 
patients
If 0 DLTs, treat all 
patients with the 
following regimen:
DKN-01 Dose Level 1 
and Nivolumab IV, both 
on
Day 1 and 15 of each 28 
day cycle
If 2 or 3 DLTs, treat all 
patients with the 
following regimen:
DKN-01 Dose Level -1 
Nivolumab IV, both on
Day 1 and 15 of each 
28 day cycle
If 0 DLTs, treat remainder of 
patients with following 
regimen: 
DKN-01 Dose Level 1 and 
Nivolumab IV,both on
Day 1 and 15 of each 28 day 
cycle
If >1 DLT, treat remainder of 
patients with following 
regimen:
DKN-01 Dose Level -1 
Nivolumab IV, both on
Day 1 and 15 of each 28 day 
cycle
Eligibility:

Histologically confirmed biliary
tract cancer

Progressed on ≥ 1 line systemic
therapy for advanced disease

Measurable disease by CT/MRI

ECOG PS 0-1


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Contents
SCHEMA.........................................................................................................................................2
1.
OBJECTIVES......................................................................................................................5
1.1
Study Design............................................................................................................5
1.2
Primary Objectives...................................................................................................5
1.3
Secondary Objectives...............................................................................................5
2.
BACKGROUND .................................................................................................................6
2.1
Biliary Tract Cancer.................................................................................................6
2.2
DKN-01 and Nivolumab..........................................................................................6
2.3
Rationale ................................................................................................................17
2.4
Correlative Studies Background ............................................................................19
3.
PARTICIPANT SELECTION...........................................................................................20
3.1
Eligibility Criteria..................................................................................................20
3.2
Exclusion Criteria ..................................................................................................21
3.3
Inclusion of Women and Minorities ......................................................................24
4.
REGISTRATION ..............................................................................................................24
4.1
General Guidelines for DF/HCC Institutions ........................................................24
4.2
Registration Process for DF/HCC Institutions.......................................................24
4.3
General Guidelines for Other Investigative Sites ..................................................25
4.4
Registration Process for Other Investigative Sites.................................................25
5.
TREATMENT PLAN........................................................................................................25
5.1
Treatment Regimen................................................................................................25
5.2
Pre-Treatment Criteria ...........................................................................................26
5.3
Agent Administration.............................................................................................27
5.4
Definition of Dose-Limiting Toxicity (DLT) ........................................................28
5.5
General Concomitant Medication and Supportive Care Guidelines......................29
5.6
Criteria for Taking a Participant Off Protocol Therapy.........................................31
5.7
Duration of Follow Up...........................................................................................32
5.8
Criteria for Taking a Participant Off Study ...........................................................32
6.
DOSING DELAYS/DOSE MODIFICATIONS ...............................................................33
6.1
General Guidelines for dose delays and modifications..........................................33
6.2
Dose Levels of DKN-01 and Nivolumab...............................................................34
6.3
Hematologic Toxicity Dose Adjustments..............................................................34
6.4
DKN-01 Dose Adjustments for Non-hematologic toxicities.................................35
6.5
DKN-01 Management for Non-hematologic toxicities .........................................36
6.6
Management of nivolumab specific adverse events ..............................................38


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7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................53
7.1
Expected Toxicities................................................................................................60
7.2
Adverse Event Characteristics...............................................................................64
7.3
Adverse Event Reporting.......................................................................................64
7.4
Reporting to the Food and Drug Administration (FDA) .......................................65
7.5
Reporting to Hospital Risk Management...............................................................65
7.6
Routine Adverse Event Reporting .........................................................................65
8.
PHARMACEUTICAL INFORMATION..........................................................................65
8.1
DKN-01..................................................................................................................66
8.2
Nivolumab..............................................................................................................69
9.
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................73
9.1
Biomarker Studies..................................................................................................73
9.2
Laboratory Correlative Studies..............................................................................74
STUDY CALENDAR ...................................................................................................................78
10.
MEASUREMENT OF EFFECT .......................................................................................80
10.1
Antitumor Effect – Solid Tumors ..........................................................................80
11.
DATA REPORTING / REGULATORY REQUIREMENTS...........................................86
11.1
Data Reporting.......................................................................................................86
11.2
Data Safety Monitoring..........................................................................................87
11.3
Collaborative Agreements Language N/A.............................................................87
12.
STATISTICAL CONSIDERATIONS...............................................................................87
12.1
Study Design/Endpoints ........................................................................................87
12.2
Sample Size, Accrual Rate and Study Duration ....................................................88
12.3
Stratification Factors..............................................................................................89
12.4
Interim Monitoring Plan ........................................................................................89
12.5
Analysis of Primary Endpoints..............................................................................89
12.6
Analysis of Secondary Endpoints..........................................................................89
12.7
Reporting and Exclusions ......................................................................................89
13.
PUBLICATION PLAN .....................................................................................................90
APPENDIX A
PERFORMANCE STATUS CRITERIA ..................................................94
APPENDIX B BIOASSAY TEMPLATES...................................................................................95
Test and Control Articles...................................................................................................95
Experimental Methods.......................................................................................................95


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1. OBJECTIVES
1.1
Study Design
This study is a multicenter, open-label phase II trial evaluating DKN 01 and Nivolumab in 
previously treated patients with advanced biliary tract cancer (BTC). Patients will be enrolled in 
a single arm trial and treated with both DKN-01 and Nivolumab IV every 2 weeks, with a safety 
lead-in period. The first three patients on trial will be treated with DKN-01 at 600mg IV and 
Nivolumab 240mg IV every 2 weeks. If no DLTs are experienced, the remainder of patients on 
trial will be treated with DKN-01 at a dose of 600mg IV. If one patient meets criteria for a dose 
limiting toxicity (DLT), three additional patients will be enrolled on study. If no additional 
patients have a DLT, DKN-01 600mg IV will be used for remainder of patients on trial.  If 2 of 
the first 3 patients or 2 of the total 6 patients have a DLT, the dose will be reduced to DKN-01 
300mg IV for the remainder of the trial. Participants that are registered for the trial but do not 
begin treatment will be replaced. Tumor response will be assessed radiographically every 8 
weeks by RECIST v1.1 criterion. Participants will receive treatment until disease progression, 
unacceptable toxicity, or death. Treatment beyond RECIST-defined progression will be 
permitted if the participant is deriving ongoing clinical benefit. No enrichment strategy will be 
used.  However, we will do a pre-planned post-hoc subgroup analysis by tumor DKK1 and PD-
L1 expression status on archival or fresh biopsy tissue. A Simon 2 stage design will be used, and 
if ≥ 1 of 13 patients respond in the first stage, the study will proceed to stage 2. 
1.2
Primary Objectives
1.2.1
Evaluate the overall response rate (ORR) of the combination of DKN-01 and nivolumab 
in patients with advanced BTC after progression on ≥1 prior systemic therapy
1.3
Secondary Objectives
1.3.1
Evaluate the safety and tolerability of the combination of DKN-01 and nivolumab in 
patients with advanced BTC
1.3.2
Evaluate the median progression free survival (PFS) and median overall survival (OS) of 
the combination of DKN-01 and nivolumab in patients with advanced BTC
1.3.3
Evaluate the ORR, PFS and OS of subgroups stratified by tumor DKK1 and PD-L1 
expression in a pre-planned post-hoc analysis
1.3.4
Explore molecular correlates of response, including circulating biomarkers, tumor tissue 
biomarkers, and stool-based gut microbiome biomarkers.


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2. BACKGROUND
2.1
Biliary Tract Cancer
Biliary tract cancer (BTC) is a rare and fatal malignancy that can involve the intrahepatic bile 
ducts, extrahepatic bile ducts, or gallbladder. Tumors of the bile ducts are more specifically 
called cholangiocarcinoma (CCA),  and the incidence of intrahepatic cholangiocarcinoma (ICC) 
is rising in the United States and globally1, 2.  While surgery can be curative if the tumor is 
detected early, recurrences and detection in the advanced stages are common. The majority of 
patients present with unresectable disease, and the 5 year survival is 10-12%. BTCs are 
biologically, histologically, and anatomically heterogenous, and the one-size-fits all approach of 
standard chemotherapy has shown limited efficacy. The current standard systemic therapy for 
advanced biliary tract cancers (ABTCs) is the combination of Gemcitabine and Cisplatin, which 
showed an 11.7 month median overall survival compared to 8.1 months with Gemcitabine 
alone3. The field is wide open for new advancements, and targeted therapies offer a promising 
approach.
Currently, there is no approved second line treatment (CT2) for advanced BTC. Regimens that 
have been studied in the first or second line setting that are currently being used in the second 
line include infusional 5-FU-based, gemcitabine-based, or oxaliplatin-based combination 
regimens. Monotherapy with infusional 5-FU, capecitabine, or gemcitabine is also sometimes 
prescribed. None of these regimens have shown superiority as second line therapy in a 
randomized study, and none of them are a clear CT2 choice based on first line data.
2.2
DKN-01 and Nivolumab
A summary of the properties of DKN-01 and nivolumab and the clinical and nonclinical 
experience with the drug are contained in the Investigator’s Brochure (IB) supplied by Leap 
Therapeutics and Bristol Myers Squibb, respectively. The IBs should be reviewed in conjunction 
with this study protocol.
2.2.1
DKN-01
DKN-01 binds to and reduces the concentration of free DKK1 in serum. A xenograft study was 
conducted in rats to study the relationship between diminished levels of free DKK1 and 
antitumor activity. The study was carried out in athymic nude female rats (strain 
Hsd:RHFoxn1rnu) bearing implanted DKK1+ human A549 NSCLC cells. Animals were treated 
with either vehicle (buffered sterile diluent) or DKN-01 (0.05, 0.2, 1.0, or 5.0 mg/kg) 
administered IV once weekly for 4 weeks. DKN-01 and DKK1 serum concentrations were 
evaluated after the first and last weekly doses and at troughs between doses. The relationship 
between drug levels, DKK1 levels, and TGI was evaluated using a PK/pharmacodynamic model. 
At the end of the study, reduction in tumor growth was observed in all active treatment arms and 
reached statistical significance in the lowest and highest DKN-01 dose-treated groups. The 
greatest mean TGI observed was 44%. Moreover, two rats exhibited complete disappearance of 


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the tumor by the end of the study, one at 5 mg/kg DKN-01 and the other at the 0.05 mg/kg 
DKN-01 dose level.
A PK/pharmacodynamic model with target-mediated drug disposition adequately described the 
relationship between total DKN-01 concentration, free DKK1 concentration, and TGI in 
xenograft-bearing nude rats. Model-predicted free DKK1 concentrations were related 
quantitatively to TGI, with 50% maximal TGI achieved at approximately 50% reduction in free 
DKK1 serum levels.
A similar PK/pharmacodynamic model was used to simulate human clinical DKN-01 
concentrations and the resulting free DKK1 serum concentrations after IV drug administration 
once every 2 weeks (Q2W).  These simulations focused on the maximal concentration for free 
DKK1 (DKK1 Cmax) over the Q2W dosing interval.  The initial baseline value of free DKK1 was 
approximately 5.4 ng/mL (data not shown).  The graphs below demonstrate that DKK1 Cmax is 
substantially reduced (relative to baseline) by the administration DKN-01.  These plots illustrate 
that in the majority of patients (> 95%), DKK1 Cmax concentrations could be suppressed to 1.5 
ng/mL or less after one dose of 300 mg DKN-01 (Figure 1). In addition, after repeat 300 mg 
Q2W dosing, the highest free DKK1 Cmax levels declined to approximately 0.76 ng/mL or less 
for 95% of patients (Figure 1). Simulations showed that when the DKN-01 dose was increased to 
600 mg, the highest free DKK1 Cmax was decreased to approximately 0.8 ng/mL after the first 
dose, and then to 0.4 ng/mL after repeat Q2W dosing to steady-state (Error! Reference source 
not found.). 
Figure 1:
Predicted Maximal Free Serum DKK1 (Cmax) Concentrations after 300 mg DKN-01 
IV Dosing Administered Once Every Two Weeks (Q2W)
Predose Week 1
Predose Week 5
Predose Week 9
Predose Week 13
Predose Week 17
Predose Week 21
Predose Week 25
0.0
0.5
1.0
1.5
Free DKK1 Cmax  (ng/mL)
0.76 ng/mL
1.5 ng/mL
Dose = 300 mg Q2W


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*Plot whiskers represent an interval containing 80% of patients.
Figure 2:
Predicted Maximal Free Serum DKK1 (Cmax) Concentrations after 600 mg DKN-01 
IV Dosing Administered Once Every Two Weeks (Q2W)*
Predose Week 1
Predose Week 5
Predose Week 9
Predose Week 13
Predose Week 17
Predose Week 21
Predose Week 25
0.0
0.5
1.0
Free DKK1 Cmax  (ng/mL)
0.4 ng/mL
0.8 ng/mL
Dose = 600 mg Q2W
*Plot whiskers represent an interval containing 90% of patients.
Several ongoing clinical studies have been conducted to determine the effect of DKN-01 on a 
variety of cancers. In Study DEK-DKK1-P102 (P102; NCT02013154), DKN-01 was dosed in 
combination with paclitaxel as well as in a separate monotherapy substudy. In this study, patients 
were administered DKN-01 at doses of 150 or 300 mg Q2W over a 28-day cycle. Patients in 
Study DEK-DKK1-P103 (P103; NCT02375880) were administered DKN-01 at doses of 150 or 
300 mg in combination with gemcitabine and cisplatin on Days 1 and 8 of each 21-day cycle. 
In both of these studies, PK/Pharmacodynamic modeling showed a dose-dependent decrease in 
free DKK1 concentrations.  In P102 and P103 the median free DKK1 Cmax was 0.39 ng/mL and 
0.50 ng/mL (respectively), and the highest individual free DKK1 Cmax was 0.86 and 0.77 ng/mL, 
respectively.  In non-responders, the median Cmax was 0.65 and 0.61 ng/mL (respectively) and 
the highest individual free Cmax observed was 5.7 and 10.8 ng/mL, respectively.  Thus, for 
patients showing a partial response, maximal free DKK1 Cmax levels did not exceed 0.86 ng/mL 
and the median free Cmax values were lower than those for the non-responders.  Currently, it is 
not understood if the 0.86 ng/mL threshold is a meaningful target for efficacy, or if median free 
DKK1 Cmax values (equal to or lower than 0.39-0.50 ng/mL) are important.  However, it can be 
stated that there is an overall trend for lower free DKK1 levels in partial responders versus non-


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responders.  By extension, higher doses of DKN-01 (i.e., 600 mg) should lower free DKK1 
levels sufficiently to see additional efficacy in patients.  
Safety data for DKN-01 are available from 30 healthy subjects treated with a single dose of 
DKN-01 monotherapy and 229 patients with cancer treated with DKN-01, of whom 65 received 
DKN-01 monotherapy; 7 received DKN-01 in combination with len/dex; 69 received DKN-01 in 
combination with paclitaxel; 51 patients received DKN-01 in combination with gem/cis; and 37 
received DKN-01 in combination with pembrolizumab.
Study DEK-DKK1-P100 was a 2-part (Parts A and B), Phase 1, multicenter, nonrandomized, 
open-label, multiple-dose, dose-escalation study of DKN-01 administered IV to patients 30 years 
of age or older. Study Part A (dose escalation) consisted of a standard 3 + 3 dose escalation 
designed to determine the MTD and DLTs of DKN-01 in patients with MM or advanced solid 
tumors. Study Part B (dose confirmation) was designed to administer DKN-01 at the MTD (or 
highest dose tested if the MTD is not reached) to further characterize safety and tolerability and 
to evaluate progression-free survival in patients with relapsed/refractory NSCLC. 
A total of 32 patients were enrolled in the study and received study treatment. Thirteen patients 
were enrolled and treated in Part A and 19 patients in Part B. In Part A, DKN-01 doses of 75, 
150, and 300 mg were administered IV QW and 600 mg was administered IV Q2W to different 
cohorts of patients. There were no DLTs observed in Part A; therefore, study Part B proceeded 
with enrolling a total of 19 patients who received 300 mg DKN-01 Q2W.
DKN-01 given IV at escalating doses from 75 mg QW to 600 mg Q2W in Part A and as a single 
IV agent at a dose of 300 mg Q2W in Part B was safe and well-tolerated. 
Twenty-nine (90.6%) of 32 patients experienced at least one TEAE during the study. The 
incidence of TEAEs was similar across dose groups (100% in all dose groups in Part A and 
84.2% in the 300 mg Q2W group in Part B). The most common types of TEAEs were 
gastrointestinal disorders (53.1%) and general disorders and administration site conditions 
(46.9%), primarily fatigue/asthenia. The most common TEAEs (those experienced by ≥10% of 
total patients) were nausea (31.3%), fatigue (28.1%), decreased appetite (25.0%), dyspnea and 
vomiting (21.9% each), constipation (18.8%), and asthenia, dysphagia, hypokalemia, pneumonia 
and productive cough (12.5% each).
Overall, 46.9% of patients experienced at least 1 treatment-related TEAE, with the most 
frequently reported treatment-related TEAEs being fatigue (25.0%), nausea (9.4%), and asthenia 
and dysgeusia (each 6.3%). 
All treatment-related TEAEs were Grade 1 or 2 in severity. There were no related SAEs and no 
discontinuations due to a TEAE.
Study DEK-DKK1-P102 is an ongoing Phase 1 non-randomized, dose-escalating, open label, 
multi-center study conducted in multiple parts (Parts A through F). In addition, a separate 


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Monotherapy Substudy is being conducted concurrently with Parts B through F. A maximum of 
approximately 224 patients aged 18 years or older with histologically confirmed recurrent or 
refractory esophageal, gastro-esophageal junction or gastric cancer with progressive disease 
requiring therapy will be enrolled in the study.
As of 20 August 2018, 124 patients have been enrolled, including 9 patients in Part A; 20 in Part 
B; 25 in Part C, 3 in Part D; 2 in Part E; 37 in Part F; and 28 in the Monotherapy Substudy. 
Overall, 3 patients have received DKN-01 150 mg+paclitaxel; 56 have received DKN-01 300 
mg+paclitaxel; 2 have received DKN-01 150 mg+pembrolizumab; 35 have received DKN-01 
300 mg+pembrolizumab; and 28 patients have received DKN-01 300 mg as monotherapy in this 
study.
Among patients treated with DKN-01 as monotherapy (N=28) in the Monotherapy Substudy, the 
most common type of TEAEs were gastrointestinal disorders (18 patients; 64%) and the most 
common individual TEAEs were fatigue (13 patients; 46%), vomiting (8 patients; 29%), 
dehydration (7 patients; 25%), and nausea, constipation, and anemia (each 6 patients; 21%).
At least 1 TEAE was considered by the Investigator to be DKN-01-related for 16 (57%) patients, 
with the most common such events being fatigue (7 patients; 25%), anemia and vomiting (each 4 
patients; 14%), and nausea and decreased appetite (each 3 patients; 11%). All other DKN-01-
related TEAEs were reported for ≤2 patients.
One (4%) patient treated with DKN-01 monotherapy experienced DKN-01-related Grade 3 
TEAEs, hyponatremia and lymphopenia. All other DKN-01-related TEAEs were Grade 1 or 2 in 
intensity.
DKN-01 combined with paclitaxel in esophagogastric cancers
Among patients treated with DKN-01 300 mg+paclitaxel (N=56), the most common type of 
TEAEs were gastrointestinal disorders (41 patients; 73%) and the most common individual 
TEAEs were fatigue (29 patients; 52%), anemia (27 patients; 48%), alopecia and peripheral 
sensory neuropathy (each 19 patients; 34%), neutropenia (17 patients; 30%), cough (16 patients; 
29%), and dyspnea (14 patients; 25%).
Among the 3 patients treated with the lower DKN-01 dose of 150 mg+paclitaxel, the TEAE 
profile was similar to that seen with the 300 mg dose, with the most common TEAEs being 
diarrhea and fatigue (each 3 patients; 100%) and arthralgia, headache, and toothache (each 2 
patients; 67%). All other TEAEs were reported for 1 patient only at this dose level.
Thirty-two (57%) of 56 patients treated with DKN-01 300 mg +paclitaxel experienced a DKN-
01-related TEAE, most commonly fatigue (13 patients; 23%), diarrhea (7 patients; 13%), nausea 
(6 patients; 11%), anemia and decreased appetite (each 5 patients; 9%), and constipation and 
neutropenia (each 4 patients; 7%).
Six (11%) patients experienced a DKN-01-related Grade 3 TEAE, including hypophosphatemia 
(2 patients; 4%), anemia, monocytosis, neutropenia, and peripheral neuropathy (1 patient; 2%). 
DKN-01-related TEAEs reported with DKN-01 150 mg + paclitaxel included single incidences 
of dysgeusia, fatigue, headache, peripheral sensory neuropathy, and stomatitis, with fatigue and 
peripheral sensory neuropathy being Grade 3 in intensity.


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Among the 56 patients who received DKN-01 300 mg + paclitaxel, 22 (39%) experienced at 
least 1 SAE, with infections and infestations being the most common type (9 patients; 16%). 
Individual SAEs reported for >1 patient included pneumonia (4 patients; 7%), and aspiration, 
lung infection, and pulmonary embolism (each 2 patients; 4%). All SAEs were considered by the 
Investigator to be unrelated to DKN-01.
Three patients experienced a Grade 5 TEAE, including aspiration (2 patients) and pulmonary 
embolism and respiratory failure in 1 patient each. For all 3 patients, the TEAE resulting in death 
was considered by the Investigator to be unrelated to DKN-01.
DKN-01 combined with pembrolizumab in esophagogastric cancers
Among patients treated with DKN-01 300 mg+pembrolizumab (N=35), the most common type 
of TEAEs were gastrointestinal disorders (18 patients; 51%) and the most common individual 
TEAEs were fatigue (11 patients; 31%), and hyponatremia, blood alkaline phosphatase 
increased, and aspartate aminotransferase increased (each 8 patients; 23%). 
Only 2 patients were treated with DKN-01 150 mg+pembrolizumab. (Two patients intended to 
be treated with DKN-01 150 mg were inadvertently treated with 300 mg DKN-01 due to site 
error; neither patient experienced a TEAE.) All TEAEs in this arm have occurred in 1 patient 
only; no individual TEAE occurred in both patients in this treatment arm.
Note that no immune system disorders or infusion-related reactions have been reported with 
DKN-01 in combination with pembrolizumab.
Fourteen (40%) patients receiving DKN-01 300 mg + pembrolizumab experienced at least 1 
TEAE considered by the Investigator to be at least possibly related to DKN-01. With the 300 mg 
dose, the most common DKN-01-related TEAEs were AST increased (5 patients; 14%), fatigue 
(4 patients; 11%), and lymphopenia (3 patients; 9%); all other such events were reported for ≤2 
patients.
One (50%) patient receiving DKN-01 150 mg + pembrolizumab experienced at least 1 TEAE 
considered by the Investigator to be at least possibly related to DKN-01, dysphonia and flushing.
Three (9%) patients at the 300 mg dose level experienced a Grade 3 DKN-01-related TEAE, 
including anemia, diarrhea, and lymphopenia (each 1 patient; 3%).
Among patients who received DKN-01 300 mg + pembrolizumab, 9 (26%) experienced at least 1 
SAE, with gastrointestinal disorders being the most common type (4 patients; 11%). The only 
SAE reported for >1 patient was decreased appetite (2 patients; 6%).
One patient experienced a Grade 5 (i.e., fatal) TEAE, gastrointestinal hemorrhage, with this 
event considered by the Investigator to be unrelated to study drug.
DKN-01 in combination with Gemcitabine/Cisplatin in biliary tract cancer
Study DEK-DKK1-P103 is a Phase 1 non-randomized, dose-escalating, open-label, multi-center 
study being conducted in 2 parts (Part A and Part B). In Part A, escalating doses of DKN-01 


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were administered to different cohorts of patients to evaluate safety and DLTs and to establish 
the MTD of DKN-01 when administered in combination with gemcitabine/cisplatin.
Overall, DKN-01 has been well tolerated and the MTD of DKN-01 in combination with 
gemcitabine and cisplatin was the highest dose tested, 300 mg, as there were no dose limiting 
toxicities in Part A of the study. However, two patients enrolled into Part B expansion and dosed 
at the MTD (300 mg) experienced DLT-equivalent toxicities including Grade 4 febrile E. Coli 
sepsis (Patient 104-013: Day 20) and Grade 3 hyperbilirubinemia (Patient 101-005: Day 106). 
All 51 (100%) patients experienced at least 1 TEAE. Overall, the most common TEAEs were 
thrombocytopenia (37 patients; 73%), neutropenia (36 patients; 71%), anemia (31 patients; 
61%), leukopenia and fatigue (each 29 patients; 57%), and aspartate aminotransferase increased, 
blood alkaline phosphatase increased, and nausea (each 28 patients; 55%).
Overall, at least 1 TEAE was considered by the Investigator to be DKN-01 related for 44 (86%) 
of patients, with the most common such events including neutropenia (27 patients; 53%), fatigue 
and thrombocytopenia (each 25 patients; 49%), leukopenia and nausea (each 19 patients; 37%), 
AST increased (18 patients; 35%), and anemia (17 patients; 33%).
For 46 (90%) of 51 patients, at least 1 TEAE was considered by the Investigator to be ≥Grade 3 
in intensity, with the most common type of such events being hematologic abnormalities within 
the blood and lymphatic system disorders SOC (38 patients; 75%), with the most common 
individual ≥Grade 3 TEAEs being neutropenia (30 patients; 59%), thrombocytopenia (17 
patients; 33%), and leukopenia (13 patients; 26%).
For 32 (63%) patients, a ≥Grade 3 was considered by the Investigator to be DKN-01-related, 
most commonly neutropenia (24 patients; 47%), thrombocytopenia (11 patients; 22%), 
leukopenia (6 patients; 12%), anemia (5 patients; 10%), AST increased (4 patients; 8%), and 
lymphopenia and ALT increased (each 3 patients; 6%).
Twenty-five (49%) of 51 patients experienced at least 1 SAE in Study DEK-DKK1-P103, 
including 24 (51%) of 47 patients in the 300 mg combination group and 1 (25%) of 4 patients in 
the 150 mg combination group. SAEs reported for >1 patient included neutropenia and 
thrombocytopenia (each 4 patients; 8%), cholangitis and sepsis (each 3 patients; 6%), and 
bacteremia, upper gastrointestinal hemorrhage, acute kidney injury, and pyrexia (each 2 patients; 
4%). All other SAEs were reported for 1 patient only. Six patients experienced SAEs that were 
considered at least possibly related to DKN-01, including thrombocytopenia and neutropenia 
(each 3 patients), Escherichia sepsis/sepsis (2 patients), and failure to thrive, leukopenia, and 
peritonitis bacterial (each 1 patient). 
Four (8%) patients experienced a Grade 5 (i.e., fatal) TEAE, including acute kidney injury, 
nephrotic syndrome, sepsis, and shock, all of which were considered unrelated to all study drugs.
Study DEK-DKK1-P204 is an ongoing Phase 2 basket study designed to evaluate DKN-01 
activity either as monotherapy or, at the Investigator’s discretion, in combination with paclitaxel 
in patients with recurrent EEC or recurrent platinum-resistant/refractory EOC who have received 
at least one prior systemic therapy for advanced disease. This basket study will enrich for 
activating β-catenin mutation and/or Wnt signaling alterations. A maximum of 94 evaluable 


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patients aged 18 years or older with histologically confirmed recurrent EEC or recurrent 
platinum-resistant/refractory EOC with PD requiring therapy will be enrolled in the study. 
Patients who are not eligible to receive paclitaxel will be enrolled in the monotherapy group by 
primary tumor.
For detailed information related to clinical safety, please refer to the Investigator’s Brochure.
2.2.2
Nivolumab 
In clinical trials, nivolumab has demonstrated an acceptable benefit-risk across multiple tumor
types, including advanced melanoma, RCC, NSCLC, and some lymphomas. Overall, the safety
profile is quite similar across multiple tumor types and is discussed further in the sections below,
including overviews from studies CA209003 and CA20937.
CA209003 is a completed Phase 1 open label, multiple dose escalation study in 306 subjects
with select previously treated advanced solid tumors, including melanoma, RCC, NSCLC,
colorectal cancer, and hormone-refractory prostate cancer. Subjects received nivolumab at doses
of 0.1, 0.3, 1, 3 or 10 mg/kg intravenously every 2 weeks, up to a maximum of 2 years of total
therapy. A total of 306 subjects were treated with nivolumab in the dose range of 0.1 - 10 mg/kg.
No maximal tolerated dose was identified in CA209003. The incidence, severity and relationship
of AEs were generally similar across dose levels and tumor types. Nivolumab related AEs of any
grade occurred in 75.2% of subjects. Of the 306 treated subjects, 303 (99.0%) subjects have at
least 1 reported AE regardless of causality. The most frequently reported AEs were fatigue
(54.9%), decreased appetite (35.0%), diarrhea (34.3%), nausea (30.1%), and cough (29.4%).
Treatment-related AEs were reported in 230 (75.2%) of the 306 subjects. The most frequently
reported treatment-related AEs were fatigue (28.1%), rash (14.7%), diarrhea (13.4%), and
pruritus (10.5%). Most treatment-related AEs were low grade. Treatment-related high grade
(Grade 3 - 4) AEs were reported in 52 (17.0%) of subjects. The most common treatment-related
high grade AEs were fatigue (2.3%) and diarrhea (1%). Drug-related SAEs occurred in 11.5% of
subjects. Grade 3 - 4 drug-related SAEs reported in at least 2 subjects included: diarrhea
(3 subjects, 1.0%), pneumonitis (3 subjects, 1.0%), pneumonia (2 subjects, 0.7%) and lipase
increased (2 subjects, 0.7%).
Select AE categories (events with a potential inflammatory mechanism requiring more frequent
monitoring and/or unique intervention such as immunosuppressants and/or endocrine
replacement therapy) include: GI AEs, pulmonary AEs, renal AEs, hepatic AEs, skin AEs, and
endocrinopathies. In addition, select AEs include a category for infusion reactions. Each
category is composed of a discrete set of preferred terms, including those of greatest clinical
relevance. These select AEs are considered events of interest based on the mechanism of action
and were previously referred to as immune-related AEs or immune-mediated AEs. Most high
grade events resolved following the treatment guidelines for the treatment of pulmonary events,
GI events, hepatic events, renal events, and endocrine events, respectively. In conclusion, the
safety profile at 3 mg/kg (n = 54) was similar to safety profile across the dose ranges from 0.1 
mg/kg to 10 mg/kg (n = 306).


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Treatment-related AEs leading to discontinuation were reported in 32 (10.5%) of the 306 treated
subjects on CA209003. The most frequent of these were pneumonitis (8 subjects; 2.6%) and 
colitis (3 subjects; 1.0%). There were 3 (1%) drug related deaths; each occurred after
development of pneumonitis.
CA209037 is an ongoing Phase 3, open-label study of nivolumab (3 mg/kg administered by
intravenous [IV] infusion every 2 weeks [Q2W]) vs investigator’s choice therapy in subjects
with previously-treated advanced melanoma. As of 30-Apr-2014, 268 subjects have been treated
with 3 mg/kg IV nivolumab in CA209037 with safety results as outlined below (source
document interim CSR, DCN930081508)4 that are consistent with the Phase 1 experience of
CA209003.
In CA209037, nivolumab related AEs of any grade occurred in 67.5% of subjects. Of the
268 subjects treated with nivolumab, 255 (95.1%) subjects had at least 1 reported AE regardless
of causality. The most frequently reported treatment-related AEs were fatigue (25.0%), pruritus
(16.0%), diarrhea (11.2%), and nausea (9.3%). Most treatment-related AEs were low grade.
Treatment-related high grade (Grade 3 - 4) AEs were reported in 24 (9.0%) of subjects. The most
common treatment-related high grade AEs were fatigue (0.7%), anemia (0.7%), diarrhea (0.4%),
and vomiting (0.4%). Drug-related SAEs occurred in 4.5% of subjects. Grade 3 - 4 drug-related
SAEs reported in at least 2 subjects included diarrhea (2 subjects, 0.7%). In addition,
drug-related SAE of hyperglycemia occurred in 0.7%.
Select AE categories (events with a potential inflammatory mechanism requiring more frequent
monitoring and/or unique intervention such as immunosuppressants and/or endocrine
replacement therapy) are:

Skin (29.1%) including pruritus (16.0%) and rash (9.3%)

GI (11.6%) including diarrhea (11.2%) and colitis (1.1%)

Endocrine (7.8%) including hypothyroidism (7.8%) and hyperthyroidism (1.9%)

Hepatic (4.5%) including AST increased (4.1%) and ALT increased (2.6%)

Pulmonary (2.2%) including pneumonitis (1.9%)

Hypersensitivity/infusion reaction (1.9%)

Renal (1.5%) including increased creatinine (0.7%), increased urea (0.4%), and 
tubulointerstitial nephritis (0.4%).
In general, these select AEs were considered by the investigator to be related to study drug,
except for AEs in the hepatic and renal select AE categories. There were few high-grade select
adverse events (n = 20), and the majority of high-grade events (13 of 20) subsequently resolved,
including those for which immunosuppressive therapy was not initiated.
Treatment-related AEs leading to discontinuation were reported in 6 (2.2%) of the 268 treated
subjects in CA209037 including single events of colitis, pancreatitis, increased ALT, increased
lipase, autoimmune neuropathy, and demyelination. There were no deaths due to drug-related
toxicity in CA209037.
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safety profile with similar trends in AEs in the 574 subjects treated with nivolumab in these
2 studies. Additional details on the safety profile of nivolumab, including results from other
clinical studies, are also available in the BMS-936558 (nivolumab) IB.
In addition to the clinical safety data outlined above, preliminary new non-clinical safety
findings of adverse pregnancy outcomes and infant losses in the absence of overt maternal
toxicity have been reported.5 The findings of increased late stage pregnancy loss and early
infant deaths/euthanasia in nivolumab exposed pregnant monkeys suggest a potential risk to
human pregnancy if there is continued treatment with BMS-936558 (nivolumab) during
pregnancy.
Summary of Nivolumab Clinical Activity
As of July 2015, > 8,600 subjects have received nivolumab in single- or multiple-dose Phase
1/2/3 studies or studies with nivolumab in combination with other therapeutics (ipilimumab,
cytotoxic chemotherapy, anti-angiogenics, and targeted therapies).
In CA209003, the clinical activity of nivolumab was demonstrated in a variety of tumor types,
including melanoma, RCC, and NSCLC. Clinical activity was noted across a range of doses
(0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg). In CA209003, a total of 306 subjects
with melanoma, RCC, and NSCLC have been evaluated for clinical activity. A response of either
CR or PR, as determined by investigator assessed tumor evaluations based on RECIST 1.1
criteria,6 has been reported at all dose levels.
In NSCLC, the most active doses were 3 and 10 mg/kg. The overall ORR of 17% was reported
with a 48-week progression-free survival rate (PFSR) of 22% (95% CI: 15 - 30%), and a
24-month overall survival rate of 24% (95% CI: 16 - 32%). Only a single response (1/33) was
reported at 1 mg/kg. Durable responses were observed in both squamous and non-squamous
subtypes. Historically, ORR of 5% to 10% and median PFS (mPFS) of 2 to 3 months has been
reported with docetaxel treatment in previously-treated NSCLC subjects.
A complete response (CR) or partial response (PR) was reported in 31% (95% CI: 22% - 41%)
of the 107 response-evaluable subjects with melanoma treated with nivolumab monotherapy
Q2W at doses ranging from 0.1 to 10 mg/kg in CA209003. The responses were durable with a
PFSR at 24 weeks of 38% (95% CI: 28 - 47%) and OS at 24 months of 48% (95% CI: 38 - 57%).
Of the 34 response evaluable RCC subjects in CA209003, responses were reported in both the
1 mg/kg (5 of 18 subjects, 27 %) and 10 mg/kg (5 of 16 subjects, 31%) treatment groups. PFSR
at 24 weeks was 33% (95% CI: 14 - 55%) in the 1 mg/kg and 37% (95% CI: 14 - 61%) in the
10 mg/kg nivolumab treatment groups. OS at 24 months was 51% (95% CI: 30 - 64%) and 44%
(95% CI: 20 - 66) in the 1 mg/kg and 10 mg/kg groups, respectively.
In CA209037, the ongoing Phase 3 study for subjects with previously-treated advanced
melanoma of nivolumab (3 mg/kg administered by intravenous [IV] infusion every 2 weeks
[Q2W]) vs investigator’s choice therapy, as of 30-Apr-2014, 120 nivolumab-treated and
47 subjects in the investigator’s choice arm are available for determination of the ORR using 
RECIST 1.1 criteria. As determined by imaging plus clinical review by a blinded independent 


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central review (BICR), the ORR for nivolumab vs the reference arm is 31.7% vs 10.6%, 
respectively. Four subjects in the nivolumab arm had a CR, whereas no subjects in the reference 
arm had a CR. Median progression-free survival was 4.7 months (95% CI: 2.3 - 6.5) vs
4.2 months (95% CI: 2.1 - 6.3) with a 6-month PFS rate of 48% (95% CI: 38 - 56) vs 34%
(95% CI: 18 - 51) in the nivolumab (n = 122) vs reference arm (n = 60), respectively.
In addition, recent clinical trial data in MEL (CA209066), and NSCLC (CA209017 and
CA209057) have demonstrated OS benefit:

In CA209066, a Phase 3 study of 418 previously untreated metastatic melanoma 
patients without a BRAF mutation, 1 year overall survival was 72.9% (95% CI: 65.5 
to 78.9) in the nivolumab group as compared to 42.1% (95% CI: 33.0 to 50.9) in the 
dacarbazine group (P < 0.001). The median progression-free survival was 5.1 months 
in the nivolumab group versus 2.2 months in the dacarbazine group (hazard ratio for 
death or progression of disease, 0.43; 95% CI, 0.34 to 0.56; P < 0.001). The objective 
response rate was 40.0% (95% CI, 33.3 to 47.0) in the nivolumab group versus 13.9% 
(95% CI, 9.5 to 19.4) in the dacarbazine group (odds ratio, 4.06; P < 0.001). The 
survival benefit with nivolumab versus dacarbazine was observed across prespecified 
subgroups, including subgroups defined by status regarding the programmed death 
ligand 1 (PD-L1).7 Additional supportive data for the clinical activity of nivolumab in 
advanced melanoma has been shown in a cohort of 107 advanced melanoma subjects 
in an outpatient setting who enrolled between 2008 and 2012. In 107 patients with 
advanced MEL treated with nivolumab, objective responses were observed in 31% of 
patients. Median OS (62% with two to five prior systemic therapies) was 16.8 
months, and 1- and 2-year survival rates were 62% and 43%, respectively. Median 
progression-free survival was 3.7 months (95% CI, 1.9 to 9.1 months), with 1- and 2-
year progression-free survival rates of 36% (95% CI, 27% to 46%) and 27% (95% CI, 
17% to 36%), respectively.8

In CA209017, a Phase 3 trial in 272 patients with metastatic squamous NSCLC with 
progression on or after platinum-based chemotherapy, subjects were randomized to 
nivolumab 3 mg/kg IV q2 week (n = 135) or docetaxel (n = 137) at 75 mg/m2 every 3 
weeks. Median OS was 9.2 months in the nivolumab group (95% CI: 7.3 - 13.3) vs 
6.0 months in the docetaxel group (95% CI: 5.1 - 7.3) (HR of 0.59, P = 0.00025).

A Phase 3 clinical trial of nivolumab vs docetaxel in Non-squamous NSCLC 
(CA209057), nivolumab monotherapy demonstrated superior OS compared with 
docetaxel, with a clinically meaningful and statistically significant improvement 
observed (HR=0.73 [95.92% CI: 0.59, 0.89]; stratified log-rank test p-value = 
0.0015). The median overall survival was 12.2 months (95% confidence interval [CI], 
9.7 to 15.0) with nivolumab versus 9.4 months (95% CI, 8.1 to 10.7) with docetaxel. 
Results of secondary endpoints of ORR, DOR, and TTR further support the antitumor 
activity of nivolumab in this population. Pre-study (baseline) PD-L1 expression was 
predictive for benefit from nivolumab for all efficacy endpoints. Interaction p-values 
reported for PD-L1 expression subgroups by each of the pre-defined expression levels 
suggested a clinically important signal of a predictive association. In PD-L1 positive 
subjects, the nivolumab group showed improved efficacy vs docetaxel across all 
efficacy endpoints (OS, ORR, and PFS). In contrast, there were no meaningful 


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differences in efficacy between the treatment groups in the PD-L1 negative subgroups 
by any expression level.9
2.3
Rationale
Biliary tract cancer (BTC) is an aggressive malignancy with a poor prognosis, and there is 
no current standard for second or third line therapy for advanced BTC. Immunotherapy 
combination trials represent a promising strategy to address this unmet medical need given the 
critical role of the immune system in the pathogenesis of BTC. Chronic inflammation is a common 
underlying risk factor for BTC and can be due to a variety of etiologies including liver fluke 
infection, choledocholithiasis, sclerosing cholangitis, viral hepatitis, and alcohol. The success of 
immune checkpoint blockade depends on an endogenous antitumor immune response, a hallmark 
of which is the presence of tumor infiltrating lymphocytes (TILs).  TILs were identified in 
approximately half of 375 BTC cases in one study10 and 27/27 cases of CCA in another study11.  
Additionally, BTCs have been shown to suppress T cell responses in a variety of ways: myeloid-
derived suppressor cells (MDSCs) have been identified in the blood of patients with BTC12 and 
PD-L1 expression has been identified in BTC in 30-100% of samples across a variety of studies11, 
13, 14. A strategy of reactivation of T cell responses is thus a seemingly rationale therapeutic 
approach in advanced BTC.
DKN-01 is a potent humanized IgG4 monoclonal antibody with neutralizing activity 
against dickkopf-1 (DKK1), a modulator of  Wnt signaling pathway, which influences a number 
of important processes such as cell growth and differentiation and bone development and adult 
bone homeostasis15-17. In a study of 138 ICC samples, 38.4% of ICC tumor samples demonstrated 
DKK1 expression, and high DKK1 expression was associated with lymph node metastasis, 
advanced tumor stage, and lower 5 year survival rates than DKK1 negative patients18. In a phase 
Ib trial of DKN-01/Gemcitabine/Cisplatin in patients with advanced refractory BTC, the overall 
response rate (ORR) was 21.3% (10/47 evaluable patients), with all 10 patients experiencing a 
confirmed PR; all 10 of these patients were treated with DKN-01 300 mg. By tumor type, the ORR 
was 20.7% (6/29 patients) in intrahepatic CCA, 25.0% (1/4 patients) in extrahepatic CCA, and 
20.0% (3/15 patients) in gallbladder cancer. Thirty-four patients experienced a best response of 
SD; thus, the disease control rate was 93.6% (44/47 patients). Among all 51 patients, median PFS 
was 35.7 weeks (95% CI, 23.3, 43.1); 25.5% (13/51) of patients were censored in the analysis, and 
among those with intra-hepatic biliary adenocarcinoma and gallbladder cancer, median PFS was 
41.3 weeks (95% CI 19.9, 53.7) and 23.4 weeks (95% CI 8.4, 43.1), respectively. By DKN-01 
dose, median PFS was 19.2 weeks (95% CI, 5.7, 48.1) and 37.7 weeks (95% CI, 23.4, 44.7) in the 
150 mg and 300 mg groups, respectively.  Among all 51 patients, median OS was 51.1 weeks 
(95% CI, 35.9, 64.9); at the time of the final analysis, 16 (31.4%) of 51 patients were alive at last 
follow-up. By DKN-01 dose, median OS was 30.9 weeks (95% CI, 15.1, 89.3) among patients 
who received DKN-01 150 mg + gem/cis (N=4) and 53.7 weeks (95% CI, 38.9, 69.9) among 
patients who received DKN-01 300 mg + gem/cis (N=47)19. Similarly, pembrolizumab has shown 
preliminary activity in biliary tract cancer (BTC) in the KEYNOTE 028 phase I trial with patients 
with treatment refractory advanced BTC with ≥ 1% PD-L1 positivity showed a 33% clinical 
benefit rate including a 17% ORR.14
Anti-PD-1 plus DKN-01 show additive effects in inhibiting tumor growth in a B16 
syngeneic mouse model, suggesting this may be a viable clinical strategy.  Studies with DKN-01 


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in this model also suggest that its locus of action is on innate immune cells (specifically natural 
killer cells) rather than on the tumor cells or the adaptive immune response. 
Based on this rationale, the current phase II trial was designed to evaluate the activity of 
the combination of DKN-01 and nivolumab in patients with advanced BTC who have progressed 
on ≥1 line of systemic therapy. 
2.4
Correlative Studies Background
Biliary tract cancers are a molecularly heterogeneous population of tumors, and identification of 
biomarkers that predict response to therapy is urgently needed to identify the subgroups most likely 
to benefit from therapies such as the combination of DKN-01 and nivolumab. 
A) RNA and DNA analysis of Pre and post treatment Diagnostic Sample 
Fresh frozen and archived formalin fixed paraffin embedded (FFPE) material from pre and post 
treatment biopsies will be analyzed for RNA and DNA markers to predict response to therapy. 
We have developed a RNA-ISH assay applicable to FFPE sectioned slides for repeat non-coding 
RNAs that appear to be linked with immune infiltrates 20, 21. We will evaluate repeat RNAs by 
RNA in situ hybridization (RNA-ISH) and immune cells by IHC (CD8, CD163, FOXP3) 
Whole exome/whole genome DNA sequencing for recurrent genomic aberrations will be 
considered for analysis. 
B) Blood Based Biomarkers for Disease Response Monitoring
Newer “liquid biopsy” technologies have emerged in recent years including circulating tumor 
(ctDNA) 22-24, circulating tumor cells (CTCs) 25-29, and exosomes 30, 31. Each of these methods 
has different advantages and the ability to evaluate these technologies in selected patient cohorts 
for their diagnostic and predictive utility in cancer is essential.  Our group has developed mature 
technologies for each of these methods to be fully evaluated in a clinical trial. Blood samples 
will be evaluated for these markers on study to determine if they correlate with response and 
potentially predict response based on the pretreatment blood sample. 
Correlative studies showed that DKN-01 combined with chemotherapy was associated with 
transient increases in circulating immune cytokines such as plasma IFN-, IL-6, IL-8 and IL-10 
but also with decreased TNF-. A high plasma IL-6 and an increase (or no decrease) in TNF- 
associated with worse outcomes. The combined regimen was also associated with changes in 
plasma biomarkers of angiogenesis at days 8 and 15, such as increased PlGF, VEGF-D, sTIE2 
and sVEGFR1 but decreases VEGF and VEGF-C. The decreased in VEGF and increase in 
sVEGFR1 may indicate an inhibition of angiogenesis, however, plasma VEGF and VEGF-C 
levels were significantly increased after 3 weeks (1 cycle) of treatment suggesting pro-
angiogeneic activity. High pretreatment VEGF-C, and decreased sVEGFR1 at day 8 and change 
in sTIE2 at day 15 associated with better survival outcomes. Plasma HGF at all time-points (but 
not changes after treatment) were associated with worse outcomes and with PK parameter 
changes after DKN-01 plus gemcitabine and cisplatin chemotherapy. Collectively, these data 


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indicate that HGF, sVEGFR1, IL-6, and TNF- should be further evaluated as biomarkers of 
poor prognosis or treatment resistance.
C) Stool Based Gut Microbiome Biomarkers
As a critical modulator for host-tumor interactions, the gut microbiome may be a modifiable 
target for adjuvant therapy and a key marker for predicting immunotherapeutic efficacy.32, 33 We 
propose to collect stool samples longitudinally throughout the study to understand microbial 
dynamics in response to treatment and identify those microbiota that may be used to predict 
disease-free progression (response), onset of treatment-related toxicities, or progression of 
disease/death.
3. PARTICIPANT SELECTION
3.1
Eligibility Criteria 
3.1.1
Histologically confirmed intra- or extrahepatic cholangiocarcinoma or gallbladder cancer
3.1.2
Participants must have measurable disease by CT/MRI by RECIST version 1.1 criteria
3.1.3
Prior chemoembolization, radiofrequency ablation, or radiation to the liver is allowed as 
long as the patient has measurable disease outside of the treated area or measurable 
progression per RECIST v1.1 at the site of the treated area.
3.1.4
Documented progression after ≥1 line of systemic therapy for advanced BTC. Prior 
adjuvant chemotherapy qualifies as this 1 line if the last cycle of adjuvant therapy was 
completed within 6 months of radiological progression.
3.1.5
Age ≥ 18 years
3.1.6
ECOG performance status ≤1 
3.1.7
Life expectancy of greater than 3 months
3.1.8
Participants must have normal organ and marrow function as defined below:

Absolute neutrophil count ≥1,500/mcL

Absolute lymphocyte count ≥0.4 x 109/L

Platelets ≥75,000/mcL

Hemoglobin ≥ 8.0 g/dL (prior transfusions are allowed if given ≥ 7 days before testing) 

Total bilirubin < 2.0 x institutional upper limit of normal; except patients with Gilbert 
Syndrome who must have a total bilirubin level of < 3.0 x ULN

AST(SGOT)/ALT(SGPT) ≤3 × institutional upper limit of normal; < 5 x ULN in case of 
liver metastases

Creatinine  < 2.0 x institutional upper limit of normal OR Creatinine clearance ≥30 


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mL/min/1.73 m2 for participants with creatinine levels ≥ ULN

International Normalized Ratio (INR)  ≤ 1.5 x ULN unless patient is receiving anticoagulant
 therapy as long as INR is within therapeutic range of intended use of anticoagulants

Serum albumin > 2.5 g/dL
3.1.9
Subjects with hepatitis B or C are eligible to enroll if they have:

Chronic HBV infection (evidenced by a positive HBV surface antigen or HBV DNA) 
as long as they have been on antiviral therapy for ≥ 4 weeks.

Chronic or resolved HCV infection (evidenced by a detectable HCV RNA or 
antibody).  Antiviral therapy is not required for chronic HCV.
3.1.10 Women of child-bearing potential and men must agree to use adequate contraception 
according to national guidelines (hormonal or barrier method of birth control; abstinence) 
prior to study entry, for the duration of study participation, and for 5 months for women 
and 7 months for men after completion of study drug administration.  
3.1.11 Female subjects must be either of non-reproductive potential (i.e., post-menopausal by 
history: ≥ 50 years old and no menses for 1 year without an alternative medical cause; 
OR history of hysterectomy, OR history of bilateral tubal ligation, OR history of bilateral 
oophorectomy) or must have a negative serum pregnancy test upon study entry. 
3.1.12 Ability to understand and the willingness to sign a written informed consent document.
3.2
Exclusion Criteria
3.2.1
Prior DKK1 inhibitor or anti-PD-1/PD-L1 treatment
3.2.2
Participants with Child-Pugh B or C cirrhosis
3.2.3
Participants with a diagnosis of ampullary cancer
3.2.4
Treatment with any of the following within the specified time frame prior to the first dose 
of DKN-01 and nivolumab:

Any non-investigational or investigational anticancer therapy within 3 weeks or have not 
recovered from side effects of such therapy prior to treatment administration (mitomycin 
within prior 5 weeks).  For targeted therapy, 5 half-lives are sufficient, even if <3 weeks. 
Concurrent participation in an observational study may be allowed after review by the 
Principal Investigator.

Patients with locoregional therapy, e.g., transarterial chemoembolization (TACE), 
selective internal radiotherapy (SIRT), external beam radiation, or ablation within 4 
weeks

Palliative limited field radiotherapy (i.e. bone metastases) within 2 weeks

Major surgery within the previous 4 weeks (the surgical incision should be fully healed 
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3.2.5
Any condition requiring systemic treatment with either corticosteroids (> 2mg daily 
dexamethasone equivalent) or other immunosuppressive medications within 14 days of 
starting the study medications. Premedication for hypersensitivity reactions (e.g. to 
contrast for CT or gadolinium for MRI) is allowed.
3.2.6
Subjects with autoimmune disease active within the last two years including but not 
limited to Crohn’s disease, ulcerative colitis, myasthenia gravis, myositis, autoimmune 
hepatitis, systemic lupus erythematosus, rheumatoid arthritis, vascular thrombosis 
associated with antiphospholipid syndrome, Wegener’s granulomatosis, Sjögren’s 
syndrome, Guillain-Barré syndrome, multiple sclerosis, type I diabetes mellitus, 
vasculitis, or glomerulonephritis

Patients with a history of autoimmune-related hypothyroidism on a stable dose of 
thyroid replacement hormone are eligible for this study.

Patients with controlled Type I diabetes mellitus on a stable insulin regimen are 
eligible
3.2.7
History of idiopathic pulmonary fibrosis, organizing pneumonia (e.g., bronchiolitis 
obliterans), idiopathic pneumonitis, or evidence of active pneumonitis on screening chest 
CT scan. History of radiation pneumonitis/fibrosis in the radiation field is permitted.
3.2.8
History of osteonecrosis of the hip or evidence of structural bone abnormalities in the 
proximal femur on magnetic resonance imaging (MRI) scan that is symptomatic and 
clinically significant. Degenerative changes of the hip joint are not excluded.
3.2.9
Known osteoblastic bony metastasis. Screening of asymptomatic subjects without a history of metastatic 
bony lesions is not required
3.2.10 Fredericia’s corrected QT interval (QTcF) ≥ 500 ms on ECG conducted during screening
3.2.11 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to DKN-01 or Nivolumab.


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3.2.12 A serious illness or medical condition(s) including, but not limited to, the following:

Known brain metastasis (not including primary brain tumors) unless patient is 
clinically stable for ≥ 1 month without systemic corticosteroids beyond 
physiologic replacement (>10 mg prednisone daily).

Known acute systemic infection.

Myocardial infarction, severe/unstable angina, symptomatic congestive heart 
failure

New York Heart Association [NYHA] Class III or IV (see Appendix C, New 
York Heart Association [NYHA] Classification) within the previous 2 months; if 
>2 months, cardiac function must be within normal limits and the patient must be 
free of cardiac-related symptoms.

Chronic nausea, vomiting, or diarrhea considered to be clinically significant in the 
opinion of the investigator.

Congenital long QT syndrome, or any known history of torsade de pointes, or 
family history of unexplained sudden death.

Other severe acute or chronic medical or psychiatric condition or laboratory 
abnormality that in the judgment of the investigator would make the patient 
inappropriate for entry into this study.
3.2.13 Patients with a history of another primary malignancy that is currently clinically
significant, and has potential for metastases or currently requires active intervention 
(except for hormonal therapy for breast or prostate cancer).
3.2.14 Patients who received treatment with live vaccines within 30 days prior to the first dose 
of study medication. Examples of live vaccines include, but are not limited to, the 
following: measles, mumps, rubella, chicken pox, shingles, yellow fever, seasonal flu, 
H1N1 flu, rabies, BCG and typhoid vaccine.
3.2.15 .
3.2.16 Prior allogeneic stem cell or solid organ transplant.
3.2.17 Known or current evidence of HIV 
3.2.18 Pregnant or lactating female.  
3.3
Inclusion of Women and Minorities
Both men and women of all races and ethnic groups are eligible for this trial.
4. REGISTRATION 
4.1
General Guidelines for DF/HCC Institutions
Institutions will register eligible participants in the Clinical Trials Management System (CTMS) 


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OnCore. Registrations must occur prior to the initiation of any protocol-specific therapy or 
intervention. Any participant not registered to the protocol before protocol-specific therapy or 
intervention begins will be considered ineligible and registration will be denied.
An investigator will confirm eligibility criteria and a member of the study team will complete the 
protocol-specific eligibility checklist.
Following registration, participants may begin protocol-specific therapy and/or intervention. 
Issues that would cause treatment delays should be discussed with the Overall Principal 
Investigator (PI). If the subject does not receive protocol therapy following registration, the 
subject must be taken off-study in the CTMS (OnCore) with an appropriate date and reason 
entered.  
4.2
Registration Process for DF/HCC Institutions
Applicable DF/HCC policy (REGIST-101) must be followed.  
4.3
General Guidelines for Other Investigative Sites
Eligible participants will be entered on study centrally at the Massachusetts General Hospital 
Cancer Center by the Study Coordinator.  
Following registration, participants should begin protocol therapy within 7 days. Issues that 
would cause treatment delays should be discussed with the Overall PI.  If the subject does not 
receive protocol therapy following registration, the subject must be taken off-study in the CTMS 
(OnCore) with an appropriate date and reason entered.  
4.4
Registration Process for Other Investigative Sites
Not applicable.
5. TREATMENT PLAN
5.1
Treatment Regimen
DKN-01 and Nivolumab will be administered on day 1 and day 15 of each 28 day cycle. 
Treatment will be administered on an outpatient basis.  Reported adverse events and potential 
risks are described in Section 7.  Appropriate dose modifications are described in Section 6.  No 
investigational or commercial agents or therapies other than those described below may be 
administered with the intent to treat the participant's malignancy.


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Table 1:
Regimen Description
Agent
Premedications
Dose
Route
Schedule
Cycle 
Length
DKN-01
None
600mg*
IV over 30 +/- 
10 minutes
Days 1 and 
15
Nivolumab
None
240mg
IV over 30 +/- 
10 min minutes, 
after completion 
of DKN-01
Days 1 and 
15
28 days
(4 weeks)
*Dose may be reduced to 300mg depending on results of DLT lead-in period
DKN-01
DKN-01 is to be administered as IV infusion over 30 minutes.  The patient’s vital signs (heart 
rate, respiratory rate, blood pressure, and temperature) should be determined within 60 minutes 
before, during (every 15 [5] minutes), and 30 (10) minutes and 2 hours (15 minutes) after the 
infusion. No premedication is needed prior to administer DKN-01.
Nivolumab
Nivolumab is to be administered as IV infusion over 30 minutes.  At the end of the infusion, 
flush the line with a sufficient quantity of normal saline or dextrose solution.
DKN-01 will be administered first followed by Nivolumab, with a minimum of 5 minutes 
between dosing.
5.2
Pre-Treatment Criteria
Treatment may be administered after review of hematology and chemistry laboratory values, but 
thyroid function tests can be pending at the time of administration. 
5.2.1
Cycle 1, Day 1

ANC ≥ 1500/mm3 

Platelet count ≥ 75×109/L 

AST and ALT at baseline grade and ≤ 5x ULN

Total bilirubin ≤ 2x ULN
5.2.2
Subsequent Cycles 
A new cycle may be initiated for patients meeting the following criteria:

ANC ≥ 1000/mm3 

Platelet count ≥ 50×109/L 

Liver associated biochemical abnormalities (e.g., AST, ALT, bilirubin) ≤Grade 1 or 
resolved to baseline grade 


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
Any other drug-related AEs that may have occurred resolved to baseline or ≤ Grade 1 
severity.
If these conditions are not met on Day 1 of a new cycle, the subject will be evaluated weekly and 
a new cycle of treatment will not be initiated until the toxicity has resolved as described above. 
The maximum treatment delay is 12 weeks for DKN-01 and 12 weeks for nivolumab. If, in the 
opinion of the investigator, a patient is receiving clinical benefit from the combination, patients 
may continue therapy after a discussion between the investigator and the Principle Investigator 
until developing documented progressive disease or otherwise meeting criteria for treatment 
discontinuation (Section 6.6).
If, in the opinion of the Investigator, a patient is receiving clinical benefit from treatment with 
the combination of DKN-01 plus nivolumab but has a compelling clinical reason after 
completion of Cycle 1 to discontinue one drug, the patient will be permitted to continue in the 
study with the other drug alone at the discretion of the treating clinician. Patients for whom the 
investigator decides it is in their best interest to stop DKN-01 and nivolumab treatment will be 
discontinued and end of treatment procedures completed.
5.3
Agent Administration
DKN-01
The vial is manufactured to deliver 20 mg of DKN-01 and must be reconstituted with 0.9% 
Sodium Chloride Solution. The drug product does not contain antimicrobial preservative and 
must be stored in refrigerated conditions (2ºC to 8ºC).
Reconstitution of the vial with 2.2 mL of 0.9% Sodium Chloride Solution gives a clear solution 
containing 10 mg/mL DKN-01. The reconstituted vials are stable for up to 8 hours at room 
temperature (up to 30°C) or refrigerated (2 to 8C).The route of administration is IV infusion via 
syringe pump only (over a period of 30 minutes). Once the reconstituted DKN-01 has been 
removed from the vials into a syringe, it must be administered to the patient within 4 hours, 
while being stored at room temperature. Therefore, the maximum time limit from reconstitution 
to completion of administration is 12 hours. A list of infusion materials compatible with DKN-01 
for Injection is provided in the study drug guidelines and in the Pharmacy Manual (Appendix C).
Study drug will be prepared and dispensed by qualified pharmacy staff under supervision of 
pharmacists.. The pharmacy must maintain an individual record for the patient.
The clinical trial centers will keep a trial specific authorization list which determines the persons 
responsible for handling of the investigational drugs. The responsible person regarding the 
following will keep accurate records in the clinical trial centers: 
•
receipt of IMP supply from sponsor (clinical trial center, principal investigator, 
identification of IMP, Batch No, Formulation, kind and size of packaging, date of expiry, 
number of study drugs per participant, number of reserve study drugs, number of study 
drugs in total, patient identification number, date and time of receipt); location of storage 
of IMP; 


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•
dispensing of IMPs in the clinical trial center (date and time, number, batch no, patient 
identification number, volume of unused solution for injection when returning). IMPs 
will not be returned to the pharmacy, they will be immediately sent to destruction after 
use.
Nivolumab
Nivolumab 100 mg (10 mg/mL) will be packaged in an open-label fashion. Five or ten
nivolumab 10 mL vials will be packaged within a carton.
Nivolumab is to be administered as an IV infusion through a 0.2-micron to 1.2-micron pore size, 
low-protein binding (polyethersulfone membrane) in-line filter at the protocol-specified doses. It 
is not to be administered as an IV push or bolus injection. Nivolumab injection can be infused 
undiluted (10 mg/mL) or diluted with 0.9% Sodium Chloride Injection, USP or 5% Dextrose 
Injection, USP to protein concentrations as low as 0.35 mg/mL. Instructions for dilution and 
infusion of nivolumab injection may be provided in the clinical protocol, pharmacy binder, or 
pharmacy reference sheet. Care must be taken to assure sterility of the prepared solution as the 
product does not contain any antimicrobial preservative or bacteriostatic agent. Nivolumab 
infusions are compatible with polyvinyl chloride (PVC) or polyolefin containers and
infusion sets and glass bottles.
5.4
Definition of Dose-Limiting Toxicity (DLT)
Toxicities will be graded and documented according to the National Cancer Institute Common
Terminology Criteria for Adverse Events (NCI CTCAE v 5.0 guidelines).
A DLT is defined as any clinically significant adverse event (AE) occurring within the first 28
days of starting the study treatment that is considered by the investigator to be possibly related or
related to DKN-01 or nivolumab. These include the following events:

Any death not clearly due to the underlying disease or extraneous causes

Non- hematologic toxicity: 
o
Grade 3 or higher 
o
Hy's law 

Hematologic toxicity:
o
Grade 4 neutropenia or thrombocytopenia > 7 days
o
≥Grade 3 thrombocytopenia with bleeding 
o
Neutropenic fever 

Grade 3 hypersensitivity reaction to DKN-01 with premedication

Grade 4 hypersensitivity reaction to DKN-01 with or without premedication

Dosing delay greater than 28 days
 The following will not be considered a DLT:

Grade 3 nausea/vomiting or diarrhea < 72 hours with adequate antiemetic and other 
supportive care


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
Grade 3 fatigue < 1 week

≥ Grade 3 electrolyte abnormality that lasts <24 to 72 hours, is not clinically complicated, 
and resolves spontaneously or responds to conventional medical interventions 

≥ Grade 3 amylase or lipase that is not associated with symptoms or clinical 
manifestations of pancreatitis 

Alopecia

Grade 3 leukopenia that resolves to ≤ grade 2 within 14 days

Grade 4 leukopenia/lymphopenia that resolves to ≤ grade 2 within 21 days

A drug-related fever ≤ Grade 3 will not be considered a DLT.

Grade 3 hypersensitivity reaction to DKN-01 with premedication (Grade 3 
hypersensitivity reaction to DKN-01 without premedication is not considered a DLT)

Grade 4 hypersensitivity reaction to DKN-01 with or without premedication

Dosing delay greater than 28 days
Management and dose modifications associated with the above adverse events are outlined in 
Section 6.
Dose escalation will proceed within each cohort according to the following scheme.  Dose-
limiting toxicity (DLT) is defined above.
Number of Participants with DLT 
at the Initial Dose Level in first 3 
patients
Escalation Decision Rule
0 out of 3
Treat the remainder of participants at DKN-01 600mg.
1 out of 3
Three (3) additional participants will be entered at the 
600mg dose level if only 3 participants were treated 
previously at that dose. If 0 DLTs are experienced, 
continue with 600mg dose level. If 1-3 DLTs are 
experienced, treat remainder of patients at 300mg dose 
level.
2-3 out of 3
Treat the remainder of participants at DKN-01 300mg.
5.5
General Concomitant Medication and Supportive Care Guidelines
Concomitant therapy includes any prescription medications or over the counter preparations used 
by a patient after 7 days preceding the screening evaluation and prior to the end of protocol 
treatment visit.
Allowed concomitant medications:


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
Patients should receive full supportive care, including transfusions of blood and blood 
products, antibiotics, anti-emetics, etc., when appropriate. 

Hematopoetic growth factors support including erythroid (eg erythropoietin, 
darbepoetin), myeloid (eg granulocyte colony-stimulating factor, granulocyte-
macrophage colony-stimulating factor), and thrombopoetic (eg romiplostim, 
eltrombopag) growth factors are allowed.

Patients who experience infusion-associated symptoms may be treated symptomatically 
with acetaminophen, diphenhydramine, and/or ranitidine or another H2 receptor 
antagonist, as per standard practice. Serious infusion associated events manifested by 
dyspnea, hypotension, wheezing, bronchospasm, tachycardia, reduced oxygen saturation, 
or respiratory distress should be managed with supportive therapies as clinically indicated 
(e.g., supplemental oxygen and β2-adrenergic agonists).

Systemic corticosteroids and TNF- inhibitors may attenuate potential beneficial 
immunologic effects of treatment with nivolumab but may be administered at 
the discretion of the treating physician after consultation with the Principle Investigator. 
If feasible, alternatives to corticosteroids should be considered. Premedication may be 
administered for Cycles  2 at the discretion of the treating physician after consultation 
with the Principle Investigator. The use of inhaled and topical corticosteroids and 
mineralocorticoids (e.g., fludrocortisone) for patients with orthostatic hypotension or 
adrenocortical insufficiency is allowed. The use of steroids for prophylaxis for CT scans 
or MRIs is allowed.

Patients who use hormonal therapy with GnRH agonists, oral contraceptives, hormone-
replacement therapy, prophylactic or therapeutic anticoagulation therapy (such as low 
molecular weight heparin or warfarin at a stable dose level), or other allowed 
maintenance therapy should continue their use. 

Patients of reproductive potential should use highly effective means of contraception.
Allowed concomitant procedures:

Biliary drainage procedures, including but not limited to endoscopic retrograde 
cholangiopancreatography (ERCP) and transhepatic percutaneous drains, are allowed 
during trial to address biliary obstruction and biliary stent exchanges at the discretion of 
the investigator.  

Paracenteses and thoracenteses

Palliative extrahepatic or liver-directed radiation is allowed.  Both drugs should be held 
for at least 1 day prior to the start of radiation, throughout radiation, and for at least 1 day 
after completion of radiation. 

Additional procedures such as ablation and chemoembolization may be allowed for 
oligometastatic progression after discussion with the Principal Investigator of the trial
Prohibited concomitant medications:

Any concomitant therapy intended for the treatment of biliary tract cancer, whether 
health authority-approved or experimental, is prohibited. This includes but is not limited 
to the following: chemotherapy, hormonal therapy, immunotherapy, radiotherapy, 


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investigational agents, or herbal therapy.

Patients must not receive live attenuated influenza vaccine within 4 weeks prior to Cycle 
1, Day 1 or at any time during the study but may receive inactivated influenza vaccines.

Medications with a known interaction with DKN-01 or nivolumab. 

Traditional herbal medicines should not be administered because the ingredients of many 
herbal medicines are not fully studied and their use may result in unanticipated drug-drug 
interactions that may cause or confound assessment of toxicity.

Patients are not allowed to receive immunostimulatory agents, including but not limited 
to IFN-, IFN-, or IL-2, during the entire study. These agents, in combination with 
nivolumab, could potentially increase the risk for autoimmune conditions.

Patients should also not receive immunosuppressive medications, including but not 
limited to cyclophosphamide, azathioprine, methotrexate, and thalidomide. These agents 
could potentially alter the activity and the safety of nivolumab. 
5.6
Criteria for Taking a Participant Off Protocol Therapy
Duration of therapy will depend on individual response, evidence of disease progression and 
tolerance. In the absence of treatment delays due to adverse event(s), treatment may continue 
until one of the following criteria applies:

Disease progression

Intercurrent illness that prevents further administration of treatment

Unacceptable adverse event(s)

Participant demonstrates an inability or unwillingness to comply with protocol 
requirements

Participant decides to withdraw from the protocol therapy

General or specific changes in the participant's condition render the participant 
unacceptable for further treatment in the judgment of the treating investigator

Treatment delay of ≥ 12 weeks of either DKN-01 or Nivolumab or both
Participants will be removed from the protocol therapy when any of these criteria apply, and the 
relevant Off-Treatment/Off-Study information will be updated in OnCore.  The reason for 
removal from protocol therapy, and the date the participant was removed, must be documented in 
the case report form (CRF). Alternative care options will be discussed with the participant.
Treatment beyond RECIST-defined progression may be permitted after discussion between the 
investigator and the Principle Investigator. The criteria to continue treatment includes: an 
absence of clinical symptoms or signs indicating clinically significant disease progression; no 


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decline in performance status; absence of rapid disease progression or threat to vital organs or 
critical anatomical sites [e.g., CNS metastasis, respiratory failure due to tumor compression, 
spinal cord compression] requiring urgent alternative medical intervention; no significant, 
unacceptable or irreversible toxicities related to study treatment; or if the participant is deriving 
ongoing clinical benefit.  
In case of early study closure, all study participants may continue to receive study treatment as 
planned in the protocol provided that they benefit from it and all protocol-specified criteria for 
continuing study treatment are met.
5.7
Duration of Follow Up
For patients who stop treatment because of confirmed disease progression:
For 100 days after stopping protocol therapy
•
 Reporting of any AE/SAE 
Follow-up visits will be conducted every 3 months ± 7days:
•
Survival status
•
Diagnosis of subsequent progression and / or new malignancy
•
Subsequent anti-cancer therapy
For patients who stop treatment in the absence of disease progression:
For 100 days after stopping protocol therapy

Reporting of any AE/SAE
Every 12 weeks/3 months ± 7days from start of treatment until 2 years after start of treatment 
relative to the date of treatment start and then every 12 weeks/3 months ± 7days until confirmed 
progression according to RECIST  

Tumor evaluation 

Abdominal contrast enhanced (CE) CT or MRI staging and CE thoracic CT

CA 19-9

Survival status

Diagnosis of subsequent progression and / or new malignancy

Subsequent anti-cancer therapy
5.8
Criteria for Taking a Participant Off Study
Participants will be removed from study when any of the following criteria apply:

Lost to follow-up

Withdrawal of consent for data submission

Death

Hospice
The reason for taking a participant off study, and the date the participant was removed, must be 
documented in the case report form (CRF). In addition, the study team will ensure Off 


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Treatment/Off Study information is updated in OnCore in accordance with DF/HCC policy 
REGIST-101.
6. DOSING DELAYS/DOSE MODIFICATIONS
6.1
General Guidelines for dose delays and modifications
Dose delays and modifications will be made as indicated in the following table(s). The 
descriptions and grading scales found in the revised NCI Common Terminology Criteria for 
Adverse Events (CTCAE) version 5.0 will be utilized for dose delays and dose modifications. A 
copy of the CTCAE version 5.0can be downloaded from the CTEP website
 http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
Each cycle should be started with all study drugs unless one drug has been held/discontinued due 
toxicity. 
If a subject is held on day 1 of a cycle, they can be re-evaluated and treated on day 8 ± 3 days if 
they meet the resumption criteria; if they are dosed on day 8 ± 3 days, the next dose will resume 
2 weeks later.  If they do not meet criteria for treatment on day 8 ± 3 days, then day 1 will be 
considered a missed dose, and they will continue with the next dose (day 15 dose) as scheduled. 
If a subject is held on day 15 of a cycle, they can be re-evaluated and treated on day 22 ± 3 days 
if they meet the resumption criteria; if they are dosed on day 22 ± 3 days, the next dose will 
resume 2 weeks later.  If they do not meet criteria for treatment on day 22 ± 3 days, then day 15 
will be considered a missed dose, and they will continue with the next cycle as scheduled. 
Interrupting dosing of the study drug(s)should depend on the attribution, as well as on the 
severity and seriousness of the toxicity to the patient subject, which is to be determined at the 
discretion of the Investigator.  In other words, how likely the toxicity is due to a specific drug, as 
well as how severe and serious the toxicity is to the patient-subject, should inform among other 
considerations (including efficacy-safety analysis), which drug or combination of drugs should 
be held/discontinued.  
Day 1 or Day 15 of any cycle may be delayed for a maximum of 12 weeks for DKN-01 or 
nivolumab to allow patients to recover from toxicity or adverse events. A treatment delay due to 
reasons other the toxicity (ex. because of holidays, weekends, inclement weather, or other 
justifiable events) should be no more than 7 calendar days and not counted as a protocol 
violation. If a delay of therapy of more than the above mentioned days are necessary, this must 
discussed with the study principle investigator.
If a significant toxicity cannot be related to a specific drug of the combination, hold both drugs 
until resolution. Restart each drug according to its specific toxicity management guidelines.


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Dose reductions are allowed for DKN-01 but not for nivolumab (please refer to guidelines 
below). 
6.2
Dose Levels of DKN-01 and Nivolumab
Table 2a: 
Dose Levels for DKN-01 if 600mg is the phase II expansion dose
Dose Level
Dose 
Level 1 (Starting Dose)
DKN-01 600mg IV
Level -1
DKN-01 300mg IV
Level -2
DKN-01 150mg IV
Dose Levels for DKN-01 if 300mg is the phase II expansion dose
Dose Level
Dose 
Level 1 (Starting Dose)
DKN-01 300mg IV
Level -1
DKN-01 150mg IV
Level -2
DKN-01 75mg IV
Table 2b:
Dose Levels for Nivolumab
Dose Level
Dose 
Level 1 (Starting Dose)
Nivolumab 240mg IV
No dose reductions allowed for nivolumab
6.3
Hematologic Toxicity Dose Adjustments
Table 3a: Hematologic Toxicity Dose Adjustments
Neutropenia
DKN-01
Nivolumab
≤ Grade 1 
(ANC <LLN-1500/mm3)
No change in dose
No change in dose
Grade 2
(ANC <1500-1000/mm3)
No change in dose.
No change in dose. 
Grade 3
(ANC <1000-500/mm3)
Or
Grade 4 
(ANC<500mm3)
Hold until < Grade 2.  
Resume at one dose level 
lower, if indicated.**
Hold until < Grade 2.  Resume at 
same dose level, if indicated.


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Neutropenia
DKN-01
Nivolumab
Second occurrence of Grade 4
(ANC <500mm3)
Off protocol therapy
Off protocol therapy
Table 3b: Hematologic Toxicity Dose Adjustments
Thrombocytopenia
DKN-01
Nivolumab
≤ Grade 1
(Plts <LLN-75,000/mm3)
No change in dose
No change in dose
Grade 2
(Plts <75,000-50,000/mm3)
No change in dose.
No change in dose. 
Grade 3
(Plts <50,000-25,000/mm3)
Or
Grade 4 
(Plts <25,000/mm3)
Hold until ≤ Grade 2.  
Resume at one dose level 
lower, if indicated.**
Hold until ≤ Grade 2.  Resume at 
same dose level, if indicated.
Second occurrence of Grade 4
(Plts <25,000/mm3)
Off protocol therapy
Off protocol therapy
6.4
DKN-01 Dose Adjustments for Non-hematologic toxicities
Table 4: DKN-01 Dose Adjustments for Non-hematologic toxicities
Toxicity Occurrence
DKN-01
First*
Restart at dose level 1 (starting dose) after toxicity resolves to ≤Grade 
1 or baseline grade.
Second
Restart at reduced dose level -1 after toxicity resolves to ≤Grade 1 or 
baseline grade.
Third
Restart at reduced dose level -2 after toxicity resolves to ≤Grade 1 or 
baseline grade.
Fourth
Discontinue
*If the toxicity is judged to be significant in the opinion of the Investigator or the Investigator 
does not wish to dose at the same dosing level, he/she may contact the Principle Investigator to 
consider a one dose reduction level with the first occurrence of toxicity.
**If the toxicity is judged to be not drug-related in the opinion of the Investigator or the 
Investigator does not wish to reduce by one dose level, he/she may contact the Principle 
Investigator to consider proceeding at the same dose level for the first occurrence of toxicity. 
Once a patient’s DKN-01 dose has been reduced, no re-escalation to a previously received dose 
is allowed at any time during the study. Intra-patient dose escalation is not permitted at any time 
during the study.
•
For patients who experience an infusion reaction to DKN-01, all attempts should be made 
to obtain blood samples for determination of anti-DKN-01 antibody (immunogenicity 
sample) and serum DKN-01 levels as close to the onset of the event as possible, at the 
resolution of the event, and 30 days following the event or just prior to the start of new 
therapy, whichever comes first. In addition, these samples may be used for determination 


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of pharmacodynamic markers.


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6.5
 DKN-01 Management for Non-hematologic toxicities
Table 5: Management of DKN-01 related toxicities
System 
Severity
Management
Follow-up
Gastrointestinal
Grade 2 or 3 nausea, 
vomiting, or diarrhea
Withhold DKN-01
Symptoms Resolve to
≤Grade 1 or baseline 
Resume DKN-01 as per Table 
4 if symptoms have improved 
to mild severity or resolution.
Grade 4 nausea, vomiting, or 
diarrhea
Permanently discontinue 
DKN-01
 
Liver
Moderate:
If baseline AST and ALT≤ 
grade 1, then AST or ALT 
>3.0 to
<5.0 x ULN and/or
Total bilirubin >2.0 to <3.0 x 
ULN
Withhold DKN-01
Increase frequency of 
liver function test 
monitoring until 
resolution
Symptoms Resolve to
≤Grade 1 or baseline
Resume DKN- 01 as per 
Table 4 if liver function tests 
are <3.0 x ULN or return to 
BL and bilirubin is
<2.0 x ULN or returns to BL.
If AST or ALT elevation 
continues to be >5 x ULN OR 
total bilirubin > 3 x ULN, see 
below.
Severe or life-threatening:
AST or ALT >5.0 x ULN 
and/or Total bilirubin >3.0 x 
ULN
Permanently discontinue 
DKN-01
Increase frequency of 
liver function test 
monitoring until 
resolution
 
Skin


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Moderate:
Non-localized rash (diffuse, 
<50% of skin surface)
Withhold DKN-01
Administer topical 
corticosteroids if there is 
no improvement of 
symptoms within 1 week
Symptoms Resolve to
≤Grade 1 or baseline
Resume DKN- 01 as per Table 
4 if dermatitis resolves or 
improves to mild (localized) 
symptoms
If symptoms worsen, see below. 
Severe or life-threatening:
Stevens-Johnson syndrome, 
toxic epidermal necrolysis or 
rash complicated by full 
thickness dermal ulceration, or 
necrotic, bullous or 
hemorrhagic manifestations
Permanently discontinue 
DKN-01
Administer systemic 
corticosteroid therapy 
 
Neurologic
Moderate:
Moderate symptoms, 
clinically detectable with no 
impact on ADLs
Withhold DKN-01
Symptoms Resolve to
≤Grade 1 or baseline
Resume DKN- 01 as per Table 
4 when symptoms resolve or 
return to BL
If symptoms worsen, see below
Severe or life-threatening:
Severe symptoms (impact on 
ADLs) or life threatening
Permanently discontinue 
DKN-01
 
Endocrine
Fatigue
Headache
Mental status 
change
Abdominal pain
Unusual bowel 
habits
Hypotension
Abnormal 
thyroid function 
tests and/or 
serum 
chemistries
Moderate to life threatening:
Document signs and/or 
symptoms of dysfunction
Endocrinopathies requiring 
hormone replacement or 
medical intervention
AEs requiring hospitalization, 
urgent medical intervention or 
interfering with ADLs
Withhold DKN-01
Evaluate endocrine 
function
Consider radiographic 
pituitary gland imaging
Continue to assess as 
indicated
Initiate appropriate 
hormone-replacement 
therapy
Symptoms Resolve to
≤Grade 1 or baseline
Resume DKN- 01 as per 
Table 4 when patient is 
stable on hormone-
replacement therapy (as 
indicated)


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Table 6: Management of DKN-01 Infusion-related reactions
Grade
Management
Grade 1
Slow the infusion rate by 50%.
Monitor the patient for worsening of condition.
For subsequent infusions, premedicate with diphenhydramine hydrochloride 50 mg 
IV (or equivalent); additional premedication may be administered at the 
investigator’s discretion.
Grade 2
Stop the infusion.
Administer diphenhydramine hydrochloride 50 mg IV (or equivalent), 
acetaminophen 650 mg orally for fever, and oxygen.
Resume the infusion at 50% of the prior rate once the infusion reaction has resolved 
or decreased to Grade 1.
Monitor for worsening of condition.
For subsequent infusions, premedicate with diphenhydramine hydrochloride 50 mg 
IV (or equivalent); additional premedication may be administered at the 
investigator’s discretion.
Grade 3
Stop the infusion and disconnect the infusion tubing from the patient.
Administer diphenhydramine hydrochloride 50 mg IV (or equivalent), 
dexamethasone 10 mg IV (or equivalent), bronchodilators for bronchospasm, and 
other medications/treatment as medically indicated.
Grade 4
Stop the infusion and disconnect the infusion tubing from the patient.
Administer diphenhydramine hydrochloride 50 mg IV (or equivalent), 
dexamethasone 10 mg IV (or equivalent), and other medications/treatment as 
medically indicated.
Give epinephrine or bronchodilators as indicated.
Hospital admission for observation may be indicated.
Patients who have a Grade 4 infusion reaction with or without premedication will 
not receive further DKN-01 treatment, but will continue to be followed on the 
protocol.
Nivolumab
6.6
Management of nivolumab specific adverse events
Toxicities associated or possibly associated with nivolumab treatment should be managed 
according to standard medical practice. Excessive activation of the immune system is a potential 
risk associated with nivolumab and has been observed when nivolumab is used in combination 
with other immunomodulating agents. Interrupt dosing of nivolumab depending on the 
attribution of the toxicity, at the discretion of the investigator. During this time, treatment may 
continue with the other non-attributable treatment agent(s).  Additional tests, such as 
autoimmune serology or biopsies, should be used to evaluate for a possible immunogenic 
etiology. Although most immune-related adverse events observed with immunomodulatory 
agents have been mild and self-limiting, such events should be recognized early and treated 
promptly to avoid potential major complications. Discontinuation of nivolumab may not have an 
immediate therapeutic effect and in severe cases, immune-related toxicities may require acute 


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management with topical corticosteroids, systemic corticosteroids or other immunosuppressive 
agents.
The Investigator should consider the benefit-risk balance a given patient may be experiencing 
prior to further administration of nivolumab. In patients who have met the criteria for permanent 
discontinuation, resumption of nivolumab may be considered if the patient is deriving benefit 
and has fully recovered from the immune-related event. Patients can be rechallenged with 
nivolumab only after approval has been documented by both the investigator (or an appropriate 
delegate) and the Principle Investigator.
6.6.1 Gastrointestinal Toxicity
Immune-related colitis has been associated with the administration of nivolumab.
Management guidelines for diarrhea or colitis are provided in Table 7.
Table 7: Management Guidelines for Gastrointestinal Events (Diarrhea or colitis)
Event
Management
Diarrhea or 
colitis, 
Grade 1

Continue nivolumab.

Initiate symptomatic treatment.

Endoscopy is recommended if symptoms persist for  7 days.

Monitor closely.
Diarrhea or 
colitis, 
Grade 2

Withhold nivolumab for up to 12 weeks after event onset.a

Initiate symptomatic treatment.

Patient referral to GI specialist is recommended.

For recurrent events or events that persist > 5 days, initiate 
treatment with 12 mg/kg/day oral prednisone or equivalent. 

If event resolves to Grade 1 or better, resume nivolumab.b

If event does not resolve to Grade 1 or better while withholding 
nivolumab, permanently discontinue nivolumab and contact 
Principle Investigatorc
Diarrhea or 
colitis, 
Grade 3

Withhold nivolumab for up to 12 weeks after event onset.a

Refer patient to gastrointestinal specialist for evaluation and 
confirmatory biopsy.

Initiate treatment with 12 mg/kg/day intravenous 
methylprednisolone or equivalent and convert to 12 mg/kg/day 
oral prednisone or equivalent upon improvement.

If event resolves to Grade 1 or better, resume nivolumabb

If event does not resolve to Grade 1 or better while withholding 
nivolumab, permanently discontinue nivolumab and contact 
Principle Investigatorc
Diarrhea or 
colitis, 
Grade 4

Permanently discontinue nivolumab and contact Principle 
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Event
Management

Refer patient to gastrointestinal specialist for evaluation and 
confirmation biopsy.

Initiate treatment with 12 mg/kg/day intravenous 
methylprednisolone or equivalent and convert to 12 mg/kg/day 
oral prednisone or equivalent upon improvement.

If event does not improve within 48 hours after initiating 
corticosteroids, consider adding an immunosuppressive agent.

If event resolves to Grade 1 or better, taper corticosteroids over 
 1 month.
aNivolumab may be withheld for a longer period of time (i.e. > 12 weeks after event onset) 
to allow for corticosteroids (if initiated) to be reduced to  10 mg/day oral prednisone or 
equivalent. The acceptable length of the extended period of time must be agreed upon by 
the investigator and the Principle Investigator.
bIf corticosteroids have been initiated, they must be tapered over  1 month to  10 mg/day 
oral prednisone or equivalent before nivolumab can be resumed.
cResumption of nivolumab may be considered in patients who are deriving benefit and have 
fully recovered from the immune-related event. Patients can be rechallenged with 
nivolumab only after approval has been documented by both the investigator (or an 
appropriate delegate) and the Principle Investigator.
All events of diarrhea or colitis should be thoroughly evaluated for other more common 
etiologies. For events of significant duration or magnitude or associated with signs of systemic 
inflammation or acute phase reactants (e.g., increased CRP or platelet count or bandemia):
Perform sigmoidoscopy (or colonoscopy, if appropriate) with colonic biopsy with three to five 
specimens for standard paraffin block to check for inflammation and lymphocytic infiltrates to 
confirm colitis diagnosis. 
6.6.2 Hepatotoxicity
Immune-related hepatitis has been associated with the administration of nivolumab.
Eligible patients must have adequate liver function, as manifested by measurements of total 
bilirubin and hepatic transaminase, and liver function will be monitored throughout study 
treatment. Patients with right upper-quadrant abdominal pain and/or unexplained nausea or 
vomiting should have LFTs performed immediately and reviewed before administration of the 
next dose of study drug.
For patients with elevated LFTs concurrent medication, viral hepatitis, and toxic or neoplastic 
etiologies should be considered and addressed, as appropriate. 
Management guidelines for hepatic events are provided in Table 8.
Table 8: Management Guidelines for Drug- Related Hepatic Eventsa


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Event
Management
Follow up for all events
Hepatic event, 
Grade >2 
(if baseline 
AST/ALT are 
WNL)
• Delay dosing of Nivolumab 
until re-treatment criteria are 
met
• Increase frequency of 
monitoring to every 3-5 days
Hepatic event, 
Grade >3 
(if baseline 
AST/ALT are 
Grade 1)
• Delay dosing of Nivolumab 
until re-treatment criteria are 
met
• Increase frequency of 
monitoring to every 3-5 days
Hepatic event, 
AST or ALT 
at 2x baseline 
or 8x ULN 
(whichever is 
lower) 
(if baseline 
AST/ALT are 
grade 2)
• Delay dosing of Nivolumab 
until re-treatment criteria are 
met
• Increase frequency of 
monitoring to every 3-5 days
• Resume dosing when re-treatment criteria 
are met
If AST or ALT levels do not improve 
with a dose delay of 3–5 days or if levels 
worsen
• Initiate steroid therapy at 0.5–2 mg/kg/d
methylprednisolone or oral equivalent
For ALT or AST levels >8× ULN
• Initiate steroid therapy promptly at 1–2 
mg/kg/d methylprednisolone or oral 
equivalent
For all patients initiating steroids
• If AST or ALT levels do not improve 
within 3–5 days or levels worsen after the 
start of steroid therapy, discuss with 
Principal Investigator possibility of adding 
mycophenolate mofetil 1 g BID
• Consult gastroenterologist
• Taper steroids once AST or ALT levels 
have declined by 1 CTCAE grade
• Taper steroids slowly over at least 1 
monthb
• Resume immuno-oncology therapy when 
AST or ALT have returned to near baseline 
unless the criteria for permanent 
discontinuation are reachedc
LFT= liver function tests. WNL= within normal limits
aNivolumab may be withheld for a longer period of time (>12 weeks after event onset) to 
allow for corticosteroids (if initiated) to be reduced to  10 mg/day oral prednisone or 
equivalent. The acceptable length of the extended period of time must be agreed upon by 
the investigator and the Principle Investigator.
bIf corticosteroids have been initiated, they must be tapered over  1 month to  10 mg/day 
oral prednisone or equivalent before nivolumab can be resumed.
cResumption of nivolumab may be considered in patients who are deriving benefit and have 
fully recovered from the immune-related event. Patients can be rechallenged with 
nivolumab only after approval has been documented by both the investigator (or an 
appropriate delegate) and the Principle Investigator.
6.6.3 Dermatologic toxicity
Treatment-emergent rash has been associated with nivolumab. The majority of cases of rash 
were mild in severity and self-limited, with or without pruritus.
A dermatologist should evaluate persistent and/or severe rash or pruritus. A biopsy should be 


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considered unless contraindicated. Management guidelines for dermatologic events are provided 
in Table 9.
Table 9: Management Guidelines for Dermatologic Events
Event
Management
Dermatologic 
event, Grade 1 

Continue nivolumab.

Consider treatment with topical corticosteroids and/or other 
symptomatic therapy (e.g., antihistamines).
Dermatologic 
event, Grade 2 

Continue nivolumab.

Consider patient referral to dermatologist.

Initiate treatment with topical corticosteroids.

Consider treatment with higher-potency topical corticosteroids if 
event does not improve
Dermatologic 
event, Grade 3 

Withhold nivolumab for up to 12 weeks after event onset.a

Refer patient to dermatologist.

Initiate treatment with 10 mg/day oral prednisone or equivalent, 
increasing dose to 12 mg/kg/day if event does not improve 
within 4872 hours.

If event resolves to Grade 1 or better, resume nivolumab.b

If event does not resolve to Grade 1 or better, permanently 
discontinue nivolumab and contact Principle Investigator.c
Dermatologic 
event, Grade 4

Permanently discontinue nivolumab and contact Principle 
Investigator.c
aNivolumab may be withheld for a period of time beyond 12 weeks to allow for corticosteroids 
to be reduced to  10 mg/day oral prednisone or equivalent. The acceptable length of the 
extended period of time must be agreed upon by the investigator and the Principle 
Investigator.
bIf corticosteroids have been initiated, they must be tapered over  1 month to  10 mg/day 
oral prednisone or equivalent before nivolumab can be resumed.
cResumption of nivolumab may be considered in patients who are deriving benefit and have 
fully recovered from the immune-related event. Patients can be rechallenged with nivolumab 
only after approval has been documented by both the investigator (or an appropriate delegate) 
and the Principle Investigator. 
6.6.4 Endocrine toxicity (including hypophysitis)
Thyroid disorders, adrenal insufficiency, diabetes mellitus and pituitary disorders have been 
associated with the administration of nivolumab. Patients with unexplained symptoms such as 
headache, fatigue, myalgias, constipation, or mental status changes, should be investigated for 
the presence of thyroid, pituitary, or adrenal endocrinopathies. The patient should be referred to 
an endocrinologist if an endocrinopathy is suspected. Thyroid-stimulating hormone (TSH) and 
free T3 and T4 levels should be measured to determine whether thyroid abnormalities are 


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present. Pituitary hormone levels and function tests (e.g. TSH, growth hormone, luteinizing 
hormone, follicle-stimulating hormone, testosterone, prolactin, adrenocorticotropic hormone 
[ACTH] levels and ACTH stimulation test) and magnetic resonance imaging (MRI) of the brain 
(with detailed pituitary sections) may help to differentiate primary pituitary insufficiency from 
primary adrenal insufficiency
Management guidelines for endocrine events are provided in Table 10. 
Table 10: Management Guidelines for Endocrine Events
Event
Management
Asymptomatic 
hypothyroidism

Continue nivolumab.

Initiate treatment with thyroid replacement hormone.

Monitor TSH monthly
Symptomatic 
hypothyroidism

Withhold nivolumab.

Initiate treatment with thyroid replacement hormone.

Monitor TSH monthly

Consider patient referral to endocrinologist.

Resume nivolumab when symptoms are controlled and 
thyroid function is improving.
Asymptomatic 
hyperthyroidism

TSH  0.1 mU/L and  0.5 mU/L:

Continue nivolumab.

Monitor TSH every 4 weeks.

TSH  0.1 mU/L:

Follow guidelines for symptomatic hyperthyroidism.
Symptomatic 
hyperthyroidism

Withhold nivolumab.

Initiate treatment with anti-thyroid drug such as 
methimazole or carbimazole as needed.

Consider patient referral to endocrinologist.

Resume nivolumab when symptoms are controlled and 
thyroid function is improving.

Permanently discontinue nivolumab and contact 
Principle Investigator for life-threatening immune-
related hyperthyroidism.c
Symptomatic adrenal 
insufficiency, 
Grade 24 

Withhold nivolumab for up to 12 weeks after event 
onset.a

Refer patient to endocrinologist.

Perform appropriate imaging.

Initiate treatment with 12 mg/kg/day intravenous 
methylprednisolone or equivalent and convert to 
12 mg/kg/day oral prednisone or equivalent upon 
improvement.

If event resolves to Grade 1 or better and patient is 
stable on replacement therapy resume nivolumab.b 


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Event
Management

If event does not resolve to Grade 1 or better or patient 
is not stable on replacement therapy while withholding 
nivolumab, permanently discontinue nivolumab and 
contact Principle Investigatorc
Hyperglycemia, 
Grade 1 or 2 

Continue nivolumab.

Initiate treatment with insulin if needed.

Monitor for glucose control.
Hyperglycemia, 
Grade 3 or 4

Withhold nivolumab.

Initiate treatment with insulin.

Monitor for glucose control.

Resume nivolumab when symptoms resolve and 
glucose levels are stable.
Hypophysitis
pan-hypopituitarism), 
Grade 2-3 

Withhold nivolumab for up to 12 weeks after event 
onset.a

Refer patient to endocrinologist.

Perform brain MRI (pituitary protocol).

Initiate treatment with 12 mg/kg/day IV 
methylprednisolone or equivalent and convert to 
12 mg/kg/day oral prednisone or equivalent upon 
improvement.a

Initiate hormone replacement therapy if clinically 
indicated.

If event resolves to Grade 1 or better, resume 
nivolumab.b

If event does not resolve to Grade 1 or better while 
withholding nivolumab, permanently discontinue 
nivolumab and contact Principle Investigator.c

For recurrent hypophysitis, treat as a Grade 4 event.
Hypophysitis
(pan-hypopituitarism), 
Grade 4

Permanently discontinue nivolumab and contact 
Principle Investigator.c

Refer patient to endocrinologist.

Perform brain MRI (pituitary protocol).

Initiate treatment with 12 mg/kg/day IV 
methylprednisolone or equivalent and convert to 
12 mg/kg/day oral prednisone or equivalent upon 
improvement.a

Initiate hormone replacement therapy if clinically 
indicated.
TSH  thyroid-stimulating hormone; 


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Event
Management
aNivolumab may be withheld for a longer period of time (i.e., >12 weeks after event onset) 
to allow for corticosteroids (if initiated) to be reduced to  10 mg/day oral prednisone or 
equivalent. The acceptable length of the extended period of time must be agreed upon by the 
investigator and the Principle Investigator.
bIf corticosteroids have been initiated, they must be tapered over  1 month to  10 mg/day 
oral prednisone or equivalent before nivolumab can be resumed.
cResumption of nivolumab may be considered in patients who are deriving benefit and have 
fully recovered from the immune-related event. Patients can be rechallenged with nivolumab 
only after approval has been documented by both the investigator (or an appropriate 
delegate) and the Principle Investigator.
6.6.5 Pulmonary toxicity
Dyspnea, cough, fatigue, hypoxia, pneumonitis and pulmonary infiltrates have been associated 
with the administration of nivolumab. Patients will be assessed for pulmonary signs and 
symptoms throughout the study and will also have CT scan of the chest performed at every 
tumor assessment.
All pulmonary events should be thoroughly evaluated for other commonly reported etiologies 
such as pneumonia or other infection, lymphangitic carcinomatosis, pulmonary embolism, heart 
failure, chronic obstructive pulmonary disease, or pulmonary hypertension.
Management guidelines for pulmonary events are provided in Table 11.
Table 11: Management Guidelines for Pulmonary Events, Including Pneumonitis
Event
Management
Pulmonary 
event, 
Grade 1 

Continue nivolumab and monitor closely.

Re-evaluate on serial imaging.

Consider patient referral to pulmonary specialist.
Pulmonary 
event, 
Grade 2

Withhold nivolumab for up to 12 weeks after event onset.a

Refer patient to pulmonary and infectious disease specialists and 
consider bronchoscopy or BAL.

Initiate treatment with 12 mg/kg/day oral prednisone or 
equivalent.

If event resolves to Grade 1 or better, resume nivolumab.b

if event does not resolve to Grade 1 or better while withholding 
nivolumab, permanently discontinue nivolumab and contact 
Principle Investigator.c

For recurrent events, treat as a Grade 3 or 4 event.
Pulmonary 
event, 
Grade 3 or 
4

Permanently discontinue nivolumab and contact Principle 
Investigator.c

Bronchoscopy or BAL is recommended.

Initiate treatment with 12 mg/kg/day oral prednisone or 
equivalent.


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Event
Management

If event does not improve within 48 hours after initiating 
corticosteroids, consider adding an immunosuppressive agent.

If event resolves to Grade 1 or better, taper corticosteroids over  1 
month.
BAL= bronchoscopic alveolar lavage; IVIG = intravenous immunoglobulin
aNivolumab may be withheld for a longer period of time (i.e. >12 weeks after event onset) to 
allow for corticosteroids (if initiated) to be reduced to  10 mg/day oral prednisone or 
equivalent. The acceptable length of the extended period of time must be agreed upon by the 
investigator and the Principle Investigator.
bIf corticosteroids have been initiated, they must be tapered over  1 month to  10 mg/day 
oral prednisone or equivalent before nivolumab can be resumed.
cResumption of nivolumab may be considered in patients who are deriving benefit and have 
fully recovered from the immune-related event. Patients can be rechallenged with nivolumab 
only after approval has been documented by both the investigator (or an appropriate 
delegate) and the Principle Investigator. 
6.6.6 Pancreatic Toxicity
Symptoms of abdominal pain associated with elevations of amylase and lipase, suggestive of 
pancreatitis, have been associated with the administration of nivolumab. The differential 
diagnosis of acute abdominal pain should include pancreatitis. Appropriate workup should 
include an evaluation for ductal obstruction, as well as serum amylase and lipase tests.
Management guidelines for pancreatic events, including pancreatitis, are provided in Table 12.
Table 12: Management Guidelines for Pancreatic Events, Including Pancreatitis
Event
Management
Amylase and lipase 
elevation, Grade 2 or 
asymptomatic grade 3

Continue nivolumab.

Monitor amylase and lipase weekly.

For prolonged elevation (e.g.,  3 weeks), consider treatment with 10 
mg/day oral prednisone or equivalent.
Symptomatic grade 3 
amylase and/or lipase 
elevation or any grade 4 
amylase and/or lipase 
elevation

Withhold nivolumab for up to 12 weeks after event onset.a

Refer patient to gastrointestinal specialist.

Monitor amylase and lipase every other day.

If no improvement, consider treatment with 12 mg/kg/day oral 
prednisone or equivalent.

If event resolves to Grade 1 or better, resume nivolumab.b

If event does not resolve to Grade 1 or better while withholding 
nivolumab, permanently discontinue nivolumab and contact 
Principle Investigator.c

For recurrent events, permanently discontinue nivolumab and contact 
Principle Investigator.c


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Event
Management
Immune-related 
pancreatitis, Grade 2 or 3

Withhold nivolumab for up to 12 weeks after event onset.a

Refer patient to gastrointestinal specialist.

Initiate treatment with 12 mg/kg/day intravenous 
methylprednisolone or equivalent and convert to 12 mg/kg/day oral 
prednisone or equivalent upon improvement.

If event resolves to Grade 1 or better, resume nivolumabb

If event does not resolve to Grade 1 or better while withholding 
nivolumab permanently discontinue nivolumab and contact Principle 
Investigator.c

For recurrent events, permanently discontinue nivolumab and contact 
Principle Investigator.c 
Immune-related 
pancreatitis, Grade 4

Permanently discontinue nivolumab and contact Principle 
Investigator.c

Refer patient to gastrointestinal specialist.

Initiate treatment with 12 mg/kg/day intravenous 
methylprednisolone or equivalent and convert to 12 mg/kg/day oral 
prednisone or equivalent upon improvement.

If event does not improve within 48 hours after initiating 
corticosteroids, consider adding an immunosuppressive agent.

If event resolves to Grade 1 or better, taper corticosteroids over  1 
month.
aNivolumab may be withheld for a longer period of time (i.e. > 12 weeks after event onset) to allow for 
corticosteroids (if initiated) to be reduced to  10 mg/day oral prednisone or equivalent. The acceptable 
length of the extended period of time must be agreed upon by the investigator and the Principle Investigator.
bIf corticosteroids have been initiated, they must be tapered over  1 month to  10 mg/day oral prednisone 
or equivalent before nivolumab can be resumed.
cResumption of nivolumab may be considered in patients who are deriving benefit and have fully recovered 
from the immune-related event. Patients can be rechallenged with nivolumab only after approval has been 
documented by both the investigator (or an appropriate delegate) and the Principle Investigator. 
6.6.7 Ocular toxicity
An ophthalmologist should evaluate visual complaints (e.g., uveitis, retinal events). Management 
guidelines for ocular events are provided in Table 13. 
Table 13: Management Guidelines for Ocular Events
Event
Management
Ocular event, 
Grade 1 

Continue nivolumab.

Patient referral to ophthalmologist is strongly recommended.

Initiate treatment with topical corticosteroid eye drops and topical 
immunosuppressive therapy.


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Event
Management

If symptoms persist, treat as a Grade 2 event.
Ocular event, 
Grade 2

Withhold nivolumab for up to 12 weeks after event onset.a

Patient referral to ophthalmologist is strongly recommended.

Initiate treatment with topical corticosteroid eye drops and topical 
immunosuppressive therapy.

If event resolves to Grade 1 or better, resume nivolumab.b

If event does not resolve to Grade 1 or better while withholding 
nivolumab permanently discontinue nivolumab and contact 
Principle Investigator c
Ocular event, 
Grade 3 or 4

Permanently discontinue nivolumab and contact Principle 
Investigator.c

Refer patient to ophthalmologist.

Initiate treatment with 12 mg/kg/day oral prednisone or 
equivalent.

If event resolves to Grade 1 or better, taper corticosteroids over 
 1 month.
aNivolumab may be withheld for a period of time (i.e. >12 weeks after event onset) to allow 
for corticosteroids (if initiated) to be reduced to  10 mg/day oral prednisone or equivalent. 
The acceptable length of the extended period of time must be agreed upon by the investigator 
and the Principle Investigator.
bIf corticosteroids have been initiated, they must be tapered over  1 month to  10 mg/day 
oral prednisone or equivalent before nivolumab can be resumed.
cResumption of nivolumab may be considered in patients who are deriving benefit and have 
fully recovered from the immune-related event. Patients can be rechallenged with nivolumab 
only after approval has been documented by both the investigator (or an appropriate delegate) 
and the Principle Investigator.
6.6.8 Immune-Related Myocarditis
Immune-related myocarditis has been associated with the administration of nivolumab. Immune-
related myocarditis should be suspected in any patient presenting with signs or symptoms 
suggestive of myocarditis, including, but not limited to, dyspnea, chest pain, palpitations, fatigue, 
decreased exercise tolerance, or syncope. Immune-related myocarditis needs to be distinguished 
from myocarditis resulting from infection (commonly viral, e.g. in a patient who reports a recent 
history of gastrointestinal illness), ischemic events, underlying arrhythmias, exacerbation of pre-
existing cardiac conditions, or progression of malignancy.
All patients with possible myocarditis should be urgently evaluated by performing cardiac 
enzyme assessment, an ECG, a chest X-ray, an echocardiogram, and a cardiac MRI as 
appropriate per institutional guidelines. A cardiologist should be consulted. An endomyocardial 
biopsy may be considered to enable a definitive diagnosis and appropriate treatment, if clinically 
indicated.


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Patients with signs and symptoms of myocarditis, in the absence of an identified alternate 
etiology, should be treated according to the guidelines in Table 14.
Table 14: Management Guidelines for Immune-Related Myocarditis
Event
Management
Immune-related 
myocarditis, Grade 1

Refer patient to cardiologist

Initiate treatment as per institutional guidelines.
Immune-related 
myocarditis, Grade 2

Withhold nivolumab for up to 12 weeks after event onset a and 
contact EORTC Principle Investigator. 

Refer patient to cardiologist

Initiate treatment as per institutional guidelines and consider 
antiarrhythmic drugs, temporary pacemaker, ECMO, or VAD 
as appropriate.

Consider treatment with 12 mg/kg/day IV methylprednisolone 
or equivalent and convert to 12 mg/kg/day oral prednisone or 
equivalent upon improvement.a

If event resolves to Grade 1 or better, resume nivolumab.b

If event does not resolve to Grade 1 or better while withholding 
nivolumab, permanently discontinue nivolumab and contact 
Principle Investigator.c
Immune-related 
myocarditis, Grade 
3-4

Permanently discontinue nivolumab and contact Principle 
Investigator.c

Refer patient to cardiologist

Initiate treatment as per institutional guidelines and consider 
antiarrhythmic drugs, temporary pacemaker, ECMO, or VAD 
as appropriate.

Initiate treatment with 12 mg/kg/day IV methylprednisolone 
or equivalent and convert to 12 mg/kg/day oral prednisone or 
equivalent upon improvement.a,b

If event does not improve within 48 hours after initiating 
corticosteroids, consider adding an immunosuppressive agent.

If event resolves to Grade 1 or better, taper corticosteroids 
over  1 month.
ECMO = extracorporeal membrane oxygenation; VAD = ventricular assist device; 
IV  intravenous.
aNivolumab may be withheld for a longer period of time (i.e.,  12 weeks after event onset) to 
allow for corticosteroids (if initiated) to be reduced to  10 mg/day oral prednisone or equivalent. 
The acceptable length of the extended period of time must be agreed upon by the investigator and 
the Principle Investigator.
bIf corticosteroids have been initiated, they must be tapered over  1 month to  10 mg/day oral 
prednisone or equivalent before nivolumab can be resumed.


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Event
Management
cResumption of nivolumab may be considered in patients who are deriving benefit and have fully 
recovered from the immune-related event. Patients can be rechallenged with nivolumab only after 
approval has been documented by both the investigator (or an appropriate delegate) and the 
Principle Investigator.
6.6.9 Infusion-Related Reactions
No premedication is indicated for administration of Cycle 1 of nivolumab. However,patients who 
experience an infusion-related reaction with Cycle 1 of nivolumab may receive premedication 
with antihistamines or antipyretics/analgesics (e.g. acetaminophen) for subsequent infusions.
Note: Metamizole (dipyrone) is prohibited in treating nivolumab associated infusion-
related reactions, due to its potential for causing agranulocytosis.
Guidelines for medical management of infusion-related reactions during Cycle 1 are provided in 
table 15. For subsequent cycles, infusion-related reactions should be managed according to 
institutional guidelines.
Table 15: Management Guidelines for Infusion-Related Reactions
Event
Management
IRR, Grade 1 

Reduce infusion rate to half the rate being given at the time of 
event onset.

After the event has resolved, the investigator should wait for 30 
minutes while delivering the infusion at the reduced rate.

If the infusion is tolerated at the reduced rate for 30 minutes after 
symptoms have resolved, the infusion rate may be increased to the 
original rate.
IRR, Grade 2

Interrupt nivolumab infusion.

Administer aggressive symptomatic treatment (e.g., oral or IV 
antihistamine, anti-pyretic, glucocorticoids, epinephrine, 
bronchodilators, oxygen).

After symptoms have resolved to baseline, resume infusion at half 
the rate being given at the time of event onset.

For subsequent infusions, consider administration of oral 
premedication with antihistamines, anti-pyretics and/or analgesics 
and monitor closely for IRRs.
IRR, Grade 3 
or 4

Stop infusion.

Administer aggressive symptomatic treatment (e.g., oral or IV 
antihistamine, anti-pyretic, glucocorticoids, epinephrine, 
bronchodilators, oxygen).

Permanently discontinue nivolumab and contact Principle 
Investigator.a
IRR = infusion-related reaction; IV  intravenous.


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Event
Management
aResumption of nivolumab may be considered in patients who are deriving benefit and have 
fully recovered from the immune-related event. Patients can be rechallenged with nivolumab 
only after approval has been documented by both the investigator (or an appropriate delegate) 
and the Principle Investigator.
6.6.10 Neurologic Disorders
Myasthenia gravis and Guillain-Barré syndrome have been observed with single-agent 
nivolumab. Patients may present with signs and symptoms of sensory and/or motor neuropathy. 
Diagnostic work-up is essential for an accurate characterization to differentiate between 
alternative etiologies. Management guidelines for neurologic disorders are provided in Table 16.
Table 16: Management Guidelines for Neurologic Disorders
Event
Management
Immune-related 
neuropathy, 
Grade 1

Continue nivolumab.

Investigate etiology.
Immune-related 
neuropathy, 
Grade 2 

Withhold nivolumab for up to 12 weeks after event onset.a

Investigate etiology.

Initiate treatment as per institutional guidelines.

If event resolves to Grade 1 or better, resume nivolumabb

If event does not resolve to Grade 1 or better, permanently 
discontinue nivolumab and contact Principle Investigatorc
Immune-related 
neuropathy, 
Grade 3 or 4

Permanently discontinue nivolumab and contact Principle 
Investigator.c 

Initiate treatment as per institutional guidelines.
Myasthenia 
gravis and 
Guillain-Barré 
syndrome (any 
grade)

Permanently discontinue nivolumab and contact Principle 
Investigator.c

Refer patient to neurologist.

Initiate treatment as per institutional guidelines.

Consider initiation of 12 mg/kg/day oral or intravenous 
prednisone or equivalent.
aNivolumab may be withheld for a longer period of time (i.e. >12 weeks after event onset) to 
allow for corticosteroids (if initated) to be reduced to  10 mg/day oral prednisone or 
equivalent. The acceptable length of the extended period of time must be agreed upon by the 
investigator and the Principle Investigator.
bIf corticosteroids have been initiated, they must be tapered over  1 month to  10 mg/day 
oral prednisone or equivalent before nivolumab can be resumed.
cResumption of nivolumab may be considered in patients who are deriving benefit and have 
fully recovered from the immune-related event. Patients can be rechallenged with nivolumab 
only after approval has been documented by both the investigator (or an appropriate delegate) 
and the Principle Investigator. 


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6.6.11 Immune-Related Meningoencephalitis
Immune-related meningoencephalitis is an identified risk associated with the administration of 
nivolumab. Immune-related meningoencephalitis should be suspected in any patient presenting 
with signs or symptoms suggestive of meningitis or encephalitis, including, but not limited to, 
headache, neck pain, confusion, seizure, motor or sensory dysfunction, and altered or depressed 
level of consciousness. Encephalopathy from metabolic or electrolyte imbalances needs to be 
distinguished from potential meningoencephalitis resulting from infection (bacterial, viral, or 
fungal) or progression of malignancy, or secondary to a paraneoplastic process.
All patients being considered for meningoencephalitis should be urgently evaluated with a CT 
scan and/or MRI scan of the brain to evaluate for metastasis, inflammation, or edema. If deemed 
safe by the treating physician, a lumbar puncture should be performed and a neurologist should 
be consulted.
Patients with signs and symptoms of meningoencephalitis, in the absence of an identified 
alternate etiology, should be treated according to the guidelines in Table 17.


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Table 17: Management Guidelines for Immune-Related Meningoencephalitis
Event
Management
Immune-related 
meningoencephalitis, 
all grades

Permanently discontinue nivolumab and contact Principle 
Investigator.a

Refer patient to neurologist.

Initiate treatment with 12 mg/kg/day IV methylprednisolone or 
equivalent and convert to 12 mg/kg/day oral prednisone or 
equivalent upon improvement.

If event does not improve within 48 hours after initiating 
corticosteroids, consider adding an immunosuppressive agent.

If event resolves to Grade 1 or better, taper corticosteroids over  1 
month.
IV  intravenous.
aResumption of nivolumab may be considered in patients who are deriving benefit and have fully 
recovered from the immune-related event. Patients can be rechallenged with nivolumab only after 
approval has been documented by both the investigator (or an appropriate delegate) and the Principle 
Investigator.
6.6.12 Immune-Related Nephritis
Immune-related nephritis is an identified risk associated with the administration of nivolumab. 
Immune-related nephritis has to be suspected if patient shows discolored urine and 
symptomatic elevation of creatinine.
Patients with signs and symptoms of nephritis, in the absence of an identified alternate etiology 
should be treated according to the guidelines in.
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS 
DEFINITIONS
ADVERSE EVENTS
An Adverse Event (AE) is defined as any new untoward medical occurrence or worsening of a 
preexisting medical condition in a clinical investigation participant administered study drug and 
that does not necessarily have a causal relationship with this treatment. An AE can therefore be 
any unfavorable and unintended sign (such as an abnormal laboratory finding), symptom, or 
disease temporally associated with the use of investigational product, whether or not considered 
related to the investigational product.
A non-serious adverse event is an AE not classified as serious. 
The protocol must include a definition for Serious Adverse Events (SAE).
SERIOUS ADVERSE EVENTS


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A Serious Adverse Event (SAE) is any untoward medical occurrence that at any dose: 

results in death

is life-threatening (defined as an event in which the participant was at risk of death at the 
time of the event; it does not refer to an event which hypothetically might have caused death 
if it were more severe)

requires inpatient hospitalization or causes prolongation of existing hospitalization (see 
NOTE below)

results in persistent or significant disability/incapacity

is an important medical event (defined as a medical event(s) that may not be immediately 
life-threatening or result in death or hospitalization but, based upon appropriate medical and 
scientific judgment, may jeopardize the subject or may require intervention [eg, medical, 
surgical] to prevent one of the other serious outcomes listed in the definition above.) 
Examples of such events include, but are not limited to, intensive treatment in an emergency 
room or at home for allergic bronchospasm; blood dyscrasias or convulsions that do not 
result in hospitalization.) 

Suspected transmission of an infectious agent (eg, pathogenic or nonpathogenic) via the 
study drug is an SAE. 
Although pregnancy and potential drug-induced liver injury (DILI), are not always serious by 
regulatory definition, however, these events must be reported within the SAEs timeline.
Any component of a study endpoint that is considered related to study therapy should be reported 
as an SAE (eg, death is an endpoint, if death occurred due to anaphylaxis, anaphylaxis must be 
reported).
NOTE: (PI determines if this information regarding hospitalizations are considered SAEs and 
should be included in the protocol. This is supplemental information that is included in 
BMS-sponsored trials)
The following hospitalizations are not considered SAEs in BMS clinical studies: 

a visit to the emergency room or other hospital department < 24 hours, that does not result 
in admission (unless considered an important medical or life-threatening event)

elective surgery, planned prior to signing consent

admissions as per protocol for a planned medical/surgical procedure

routine health assessment requiring admission for baseline/trending of health status (eg, 
routine colonoscopy)

Medical/surgical admission other than to remedy ill health and planned prior to entry into 
the study. Appropriate documentation is required in these cases.


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
Admission encountered for another life circumstance that carries no bearing on health 
status and requires no medical/surgical intervention (eg, lack of housing, economic 
inadequacy, caregiver respite, family circumstances, administrative reason).
Admission for administration of anticancer therapy in the absence of any other SAEs (applies to 
oncology protocols)
ADVERSE EVENT Collection and REPORTING INFORMATION:

All Serious Adverse Events (SAEs) that occur following the subject’s written consent to 
participate in the study through 100 days of discontinuation of dosing must be reported to 
BMS Worldwide Safety and Leap Therapeutics, whether related or not related to study 
drug. If applicable, SAEs must be collected that relate to any later protocol-specified 
procedure (eg, a follow-up skin biopsy).
BMS Worldwide Safety Contact Information 

Worldwide.Safety@BMS.com

+1 609-818-3804
Leap Therapeutics

DKN_Safety@leaptx.com 

Safety-Inbox@novellaclinical.com

Following the subject’s written consent to participate in the study, all SAEs, whether 
related or not related to study drug, are collected, including those thought to be associated 
with protocol-specified procedures. The investigator should report any SAE occurring after 
these aforementioned time periods, which is believed to be related to study drug or 
protocol-specified procedure.

An SAE report should be completed for any event where doubt exists regarding its 
seriousness; 

If the investigator believes that an SAE is not related to study drug, but is potentially 
related to the conditions of the study (such as withdrawal of previous therapy or a 
complication of a study procedure), the relationship should be specified in the narrative 
section of the SAE Report Form.
An appropriate SAE form (e.g.  ex-US = CIOMS form or USA = Medwatch form) should be 
used to report SAEs to BMS.  If you prefer to use your own Institutional form, it must be 
reviewed by the BMS Protocol Manager prior to study initiation to ensure that at a minimum all 
of the data elements on the CIOMS form are present.  Note:  Please include the BMS Protocol 
number on the SAE form or on the  cover sheet with the SAE form transmission.  
The CIOMS form is available at: http://www.cioms.ch/index.php/cioms-form-i 
The MedWatch form is available at: MedWatch 3500 Form 


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
The Sponsor will reconcile the clinical database AE cases (case level only) transmitted to 
BMS Global Pharmacovigilance (Worldwide.Safety@bms.com). 

The Investigator will request from BMS GPV&E, 
aepbusinessprocess@bms.com the SAE reconciliation report and include 
the BMS protocol number every 3 months and prior to data base lock or 
final data summary

GPV&E will send the investigator the report to verify and confirm all 
SAEs have been transmitted to BMS GPV&E.

The data elements listed on the GPV&E reconciliation report will be used 
for case identification purposes. If the Investigator determines a case was 
not transmitted to BMS GPV&E, the case should be sent immediately to 
BMS (Worldwide.Safety@bms.com).

In addition to the Sponsor Investigator’s responsibility to report events to their local HA, 
suspected serious adverse reactions (whether expected or unexpected) shall be reported by 
BMS to the relevant competent health authorities in all concerned countries according to 
local regulations (either as expedited and/or in aggregate reports).

In accordance with local regulations, BMS will notify sponsor investigators of all reported 
SAEs that are suspected (related to the investigational product) and unexpected (ie, not 
previously described in the IB). An event meeting these criteria is termed a Suspected, 
Unexpected Serious Adverse Reaction (SUSAR). Sponsor investigator notification of these 
events will be in the form of either a SUSAR Report or a Semi-Annual SUSAR Report. 
Other important findings which may be reported by BMS as an Expedited Safety 
Report (ESR) include: increased frequency of a clinically significant expected SAE, an 
SAE considered associated with study procedures that could modify the conduct of the 
study, lack of efficacy that poses significant hazard to study subjects, clinically 
significant safety finding from a nonclinical (eg, animal) study, important safety 
recommendations from a study data monitoring committee, or sponsor or BMS 
decision to end or temporarily halt a clinical study for safety reasons.
Upon receiving an ESR from BMS, the investigator must review and retain the ESR 
with the IB. Where required by local regulations or when there is a central IRB/IEC 
for the study, the sponsor will submit the ESR to the appropriate IRB/IEC. The 
investigator and IRB/IEC will determine if the informed consent requires revision. The 
investigator should also comply with the IRB/IEC procedures for reporting any other 
safety information. 
SAEs, whether related or not related to study drug, and pregnancies must be reported to BMS 
within 24 hours \ 1 Business Day of becoming aware of the event. SAEs must be recorded on 
either CIOMS, MedWatch, or approved site SAE form.  
Pregnancies must be reported and submitted to BMS. BMS will perform due diligence follow-up 
using the BMS Pregnancy Form which the investigator must complete. 
SAE Email Address:  Worldwide.Safety@BMS.com


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SAE Facsimile Number:  +1 609-818-3804
If only limited information is initially available, follow-up reports are required. (Note: Follow-up 
SAE reports should include the same investigator term(s) initially reported.) 
If an ongoing SAE changes in its intensity or relationship to study drug or if new information 
becomes available, a follow-up SAE report should be sent within 24 hours \ 1 Business Day to 
BMS using the same procedure used for transmitting the initial SAE report. 
All SAEs should be followed to resolution or stabilization.
The causal relationship to study drug is determined by a physician and should be used to assess 
all adverse events (AE). The casual relationship can be one of the following:
Related: There is a reasonable causal relationship between study drug administration and the AE.
Not related: There is not a reasonable causal relationship between study drug administration and 
the AE. 
The term "reasonable causal relationship" means there is evidence to suggest a causal 
relationship. 
Adverse events can be spontaneously reported or elicited during open-ended questioning, 
examination, or evaluation of a subject. (In order to prevent reporting bias, subjects should not be 
questioned regarding the specific occurrence of one or more AEs.)
NONSERIOUS ADVERSE EVENT

Non-serious Adverse Events (AE) are to be provided to BMS in aggregate via interim or 
final study reports as specified in the agreement or, if a regulatory requirement [eg, IND 
US trial] as part of an annual reporting requirement. 

Non-serious AE information should also be collected from following the subject’s written 
consent to participate in the study. 
Non-serious Adverse Event Collection and Reporting
The collection of non-serious AE information should begin following the subject’s written 
consent to participate in the study. All non-serious adverse events (not only those deemed to be 
treatment-related) should be collected continuously during the treatment period and for a 
minimum of 100  days following the last dose of study treatment. 
Non-serious AEs should be followed to resolution or stabilization, or reported as SAEs if they 
become serious. Follow-up is also required for non-serious AEs that cause interruption or 
discontinuation of study drug and for those present at the end of study treatment as appropriate. 


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Laboratory Test Abnormalities
All laboratory test results captured as part of the study should be recorded following institutional 
procedures. Test results that constitute SAEs should be documented and reported to BMS as 
such.
The following laboratory abnormalities should be documented and reported appropriately:

any laboratory test result that is clinically significant or meets the definition of an SAE

any laboratory abnormality that required the participant to have study drug discontinued or 
interrupted

any laboratory abnormality that required the subject to receive specific corrective therapy. 
It is expected that wherever possible, the clinical rather than laboratory term would be used by 
the reporting investigator (eg, anemia versus low hemoglobin value).
Drug Induced Liver Injury (DILI):
Wherever possible, timely confirmation of initial liver-related laboratory abnormalities should 
occur prior to the reporting of a potential DILI event. All occurrences of potential DILIs, meeting 
the defined criteria, must be reported as SAEs.
Potential drug induced liver injury is defined as: 
1) AT (ALT or AST) elevation > 3 times upper limit of normal (ULN)
AND
2) Total bilirubin > 2 times ULN, without initial findings of cholestasis (elevated serum alkaline 
phosphatase)
AND
3) No other immediately apparent possible causes of AT elevation and hyperbilirubinemia, 
including, but not limited to, viral hepatitis, pre-existing chronic or acute liver disease, or the 
administration of other drug(s) known to be hepatotoxic.
AEs of Special Interest (Product Specific Usually a regulatory requirement.  Remove if 
not applicable)
Pregnancy
If, following initiation of the investigational product, it is subsequently discovered that a study 
participant is pregnant or may have been pregnant at the time of investigational product 
exposure, including during at least 5 half-lives after product administration, the investigational 
product will be permanently discontinued in an appropriate manner (eg, dose tapering if 
necessary for participant). 
The investigator must immediately notify Worldwide.Safety@bms.com of this event and 
complete one of the following forms within 24 hours of awareness of the event via either the 
CIOMS, MedWatch or appropriate Pregnancy Surveillance Form in accordance with SAE 
reporting procedures. 


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Protocol-required procedures for study discontinuation and follow-up must be performed on the 
participant.
Follow-up information regarding the course of the pregnancy, including perinatal and neonatal 
outcome and, where applicable, offspring information must be reported on the CIOMS, 
MedWatch, BMS Pregnancy Surveillance Form, or approved site SAE form.  A BMS Pregnancy 
Surveillance Form may be provided upon request.
Any pregnancy that occurs in a female partner of a male study participant should be reported to 
BMS. Information on this pregnancy will be collected on the Pregnancy Surveillance Form. In 
order for Sponsor or designee to collect any pregnancy surveillance information from the female 
partner, the female partner must sign an informed consent form for disclosure of this 
information. 
Other Safety Considerations
Any significant worsening noted during interim or final physical examinations, 
electrocardiograms, X-rays, and any other potential safety assessments, whether or not these 
procedures are required by the protocol, should also be recorded as a non-serious or serious AE, 
as appropriate, and reported accordingly. 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The 
following list of reported and/or potential AEs (Section 7.1) and the characteristics of an 
observed AE (Section 7.2) will determine whether the event requires expedited reporting in 
addition to routine reporting.


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7.1
Expected Toxicities 
See investigator’s brochure for DKN-01 and Nivolumab for full and up-to-date list of expected 
toxicities. 
7.1.1
Adverse Events Lists
7.1.1.1
Adverse Event List(s) for DKN-01
Risks associated with DKN-01 therapy. Events MAY BE SERIOUS (i.e. causing hospitalization, 
life-threatening, or where noted, may cause death) and include the following:
Very Common (>10%)
• Fatigue
• Nausea
Common (>1% and <10%)
• Vomiting
• Anemia (decrease in the amount of red blood cells in the blood)
• Decreased appetite
• Arthralgia (joint pain)
• Dysgeusia (changes in sense of taste)
• Hyponatremia* (decrease in blood sodium level)
• Lymphopenia* (decrease of lymphocyte concentration in blood)
• • Constipation
• 
• Leukopenia (decreased white blood cell level)
• Muscle Spasms
• • Myalgia (muscle pain)
• 
• • Oral pain
• Diarrhea (watery, loose or soft stools)
• Dizziness
• Abdominal distension (swelling) 
• Abnormal liver function test (alanine aminotransferase and/or aspartate aminotransferase 
increased) which possibly can lead to liver damage 
• Headache 
• Low levels of phosphorous in the blood (hypophosphatemia)
• Muscle weakness 
• Weakness (asthenia)
• Blood cholesterol increased 
• Chills
• Indigestion (dyspepsia) 
• Shortness of breath (dyspnea) 
• Trouble sleeping (insomnia) 
• Abnormal blood test that can indicate longer than normal blood clotting time (increased 
international normalized ratio) 


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• Excess protein in the urine (proteinuria) which may cause fluid retention
• Decreased number of platelets in the blood (thrombocytopenia). A low platelet count increases 
your risk of bleeding including: nosebleeds, bruising, stroke, and/or digestive system bleeding. If 
your platelet count it too low,  you may need a platelet transfusion. 
• 
• Stomatitis (inflammation in the mouth)
• Weight decreased
* Severe
Risks associated with DKN-01 therapy when given in combination with a PD-1 inhibitor. Events 
MAY BE SERIOUS (i.e. causing hospitalization, life-threatening, or where noted, may cause 
death), and include the following:
Very Common (>10%)

Aspartate aminotransferase increased

Fatigue

Blood alkaline phosphatase increased (an enzyme from liver and bone), which may cause 
fatigue and yellowing of the skin and eyes
Common (>1% and <10%)

Alanine aminotransferase increased

Lymphopenia* (low level of lymphocytes in blood)

Anemia

Hypocalcemia (low calcium concentration in the blood)

Nausea

Abdominal distension

Anemia

Arthralgia

Diarrhea*

Dysphonia (difficulty speaking)

Dyspnea (Shortness of breath)

Flushing

Hypoalbuminemia

Hyponatremia (low sodium level in blood)

Hypophosphatemia (low level of phosphorous in blood)

Leukopenia (decreased white blood cells)

Neuropathy peripheral

Night sweat

Decreased appetite 

Blood creatinine increased which may indicate kidney injury 

Blood bilirubin increased (an abnormal liver test) which may cause yellowing of the skin 
and/or eyes 


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
Hypokalemia (low level of potassium in the blood) 

Myalgia (muscle pain) 

Generalized itching (pruritus) 

Fever (pyrexia) 

Generalized rash

Vomiting

Abdominal pain

Weakness (asthenia)

Dehydration

Dizziness

Dysgeusia (abnormal taste or sense of taste) 

Eczema

Gamma-glutanyl transferase increased (abnormal liver test) which suggests possible liver 
damage 

Homer’s Syndrome, which may cause decreased pupil size, a dropping eyelid and 
decreased sweating on the affected side of the face

High blood sugar (hyperglycemia) 

High blood pressure (hypertension) 

Hypomagenseaemia (low levels of magnesium in blood) 

Influenza (Flu) like illness

Pink or Purple Spots on the skin (Lichen planus) 

Muscle spasms

Muscle spasticity

Musculoskeletal pain 

Neutrophil count decreased (low white blood cell count) which can increase the risk of 
infection including fever, pain, redness, and/or difficult breathing. These symptoms may 
be serious or life threatening 

Painless detachment of the nail from the nail bed (onychoclasis) 

Low blood pressure after standing up (orthostatic hypotension)

Excess fluid in the lungs (pleural effusion) 

Pneumonia 

High levels of protein in the urine (proteinuia) which may cause fluid retention

Rash that is red and may be flat and/or have bumps (Rash maculopapular)  

Mouth sores or swelling (stomatitis) 

Fainting (syncope) 

Upper-airway cough syndrome 

* Severe
7.1.1.2
Adverse Event List for Nivolumab
Nivolumab may cause the side effects listed below, severity or duration of these
side effects may vary for each participant. This information is based on data from


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subjects in other clinical trials with nivolumab. In addition, there may be side
effects that are not yet known that may occur. 
Very common side effects of nivolumab are (≥ 10%)

Diarrhea

Fatigue

Itching

Rash
Common side effects of nivolumab include (≥1 to < 10%)

Abdominal pain

Alkaline phosphatase increased: lab test result associated with liver or bone 
abnormalities

Allergic reaction/hypersensitivity

ALT increased: lab test result associated with abnormal liver function

Amylase increased: lab test result associated with pancreas inflammation

AST increased: lab test result associated with abnormal liver function

Bilirubin (liver function blood test) increased

Chills

Constipation

Cough

Creatinine increased: lab test result associated with decreased kidney Function

Decreased appetite

Dizziness or Vertigo (feeling off balance which can lead to dizziness)

Dry mouth

Dry skin

Fever

Headache

High blood pressure

Increased blood sugar

Inflammation of the colon

Inflammation of the mouth

Infusion related reaction

Joint pain or stiffness

Lipase increased: lab test result associated with pancreas inflammation

Loss of color (pigment) from areas of skin

Lung inflammation (pneumonitis - see details below)

Musculoskeletal pain

Nausea

Redness (of the skin)

Shortness of breath

Sodium levels in the blood low

Swelling, including face, arms, and legs

Thyroid gland function decreased (hypothyroidism). It is a condition where


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
your thyroid gland doesn’t produce enough hormones. The common signs

of an underactive thyroid are tiredness, weight gain and feeling

depressed.

Thyroid gland function increased (hyperthryoidism). It is a condition in

which the thyroid overproduces hormones. The common symptoms

include weight loss, weakness, irregular heartbeat, and difficulty sleeping

Tingling, burning, numbness or weakness, possibly in arms, legs, hands

and feet

Upper respiratory tract infection

Vomiting
Uncommon side effects of nivolumab include (≥0.1 to < 1%)

Adrenal gland function decreased

Bronchitis

Cranial nerve disorder

Diabetes

Dry eye

Hair loss

Heart rate increased

Heart rhythm abnormal

Hives

Inflammation of the eye

Inflammation of the kidney

Inflammation of the pancreas

Inflammation of the pituitary gland

Inflammation of the stomach

Inflammation of the thyroid gland

Liver inflammation

Low blood pressure

Lung infiltrates, associated with infection or inflammation

Pituitary gland function decreased

Psoriasis: characterized by patches of abnormal, scaly skin

Renal (kidney) failure or kidney injury

Respiratory failure

Vision blurred
Rare side effects of nivolumab include (≥0.01 to < 0.1%)

Anaphylactic reaction (severe allergic reaction)

Damage to the protective covering of the nerves in the brain and spinal

cord

Diabetes complications resulting in excess blood acids and diabetic coma

Guillain-Barre syndrome, an autoimmune disorder associated with

progressive muscle weakness or paralysis

Inflammation of blood vessels


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
Inflammation of the brain, potentially life-threatening or fatal

Inflammation of the heart

Muscle inflammation

Myasthenic syndrome (neurologic syndrome characterized by muscle

weakness) including myasthenia gravis, a nerve disease that may cause

weakness of eye, face, breathing, and swallowing muscles.

Pemphigoid: blistering of the skin or mouth caused by the immune system

attacking healthy tissue

Rhabdomyolysis: muscle fiber released into the blood stream which could

damage your kidneys

Rosacea: acne-like skin condition resulting in redness of face

Sarcoidosis, a disease involving abnormal collections of inflammatory cells

(granulomas) in organs such as lungs, skin, and lymph nodes

Stevens Johnson syndrome: inflammatory disorder of skin and mucous

membranes, resulting in blistering and shedding of skin

Toxic epidermal necrolysis: a potentially fatal disease characterized by

blistering and peeling of the top layer of skin resembling a severe burn

Histiocytic necrotizing lymphadenitis or Kikuchi lymphadenitis: disorder of

the lymph nodes which causes the lymph nodes to become enlarged,

inflamed and painful, commonly affecting lymph nodes of the neck and

possibly associated with fever or muscle and joint pains.

Vogt Koyanagi Harada syndrome; a disease that affects the pigmented

tissue; this may affect the eye leading to swelling, pain and/or blurred

vision; the ear leading to hearing loss, ringing in the ears and/or skin

leading to loss of skin color
7.2
Adverse Event Characteristics

CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
5.0 will be utilized for AE reporting.  All appropriate treatment areas should have access 
to a copy of the CTCAE version 5.0.  A copy of the CTCAE version 5.0 can be 
downloaded from the CTEP web 
sitehttps://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/CTCAE_v5
_Quick_Reference_5x7.pdf

For expedited reporting purposes only:  

AEs for the agent(s) that are listed above should be reported only if the adverse event 
varies in nature, intensity or frequency from the expected toxicity information which 
is provided.

Other AEs for the protocol that do not require expedited reporting are outlined in the 


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next section (Expedited Adverse Event Reporting) under the sub-heading of Protocol-
Specific Expedited Adverse Event Reporting Exclusions.

Attribution of the AE:
o
Definite – The AE is clearly related to the study treatment.
o
Probable – The AE is likely related to the study treatment.
o
Possible – The AE may be related to the study treatment.
o
Unlikely – The AE is doubtfully related to the study treatment.
o
Unrelated – The AE is clearly NOT related to the study treatment.
7.3
Adverse Event Reporting 
7.3.1
In the event of an unanticipated problem or life-threatening complications treating 
investigators must immediately notify the Overall PI.
7.3.2
Investigators must report to the Overall PI any adverse event (AE) that occurs after the 
initial dose of study treatment, during treatment, or within 100 days of the last dose of 
treatment on the local institutional SAE form. 
7.3.3
DF/HCC Adverse Event Reporting Guidelines
Investigative sites within DF/HCC will report AEs directly to the DFCI Office for 
Human Research Studies (OHRS) per the DFCI IRB reporting policy. 
7.3.4
Protocol-Specific Adverse Event Reporting Exclusions
None
7.4
Reporting to the Food and Drug Administration (FDA)
The Overall PI, as study sponsor, will be responsible for all communications with the FDA. The 
Overall PI will report to the FDA, regardless of the site of occurrence, any serious adverse event 
that meets the FDA’s criteria for expedited reporting following the reporting requirements and 
timelines set by the FDA.
7.5
Reporting to Hospital Risk Management
Participating investigators will report to their local Risk Management office any participant 
safety reports, sentinel events or unanticipated problems that require reporting per institutional 
policy. 
7.6
 Routine Adverse Event Reporting
All Adverse Events must be reported in routine study data submissions to the Overall PI on the 
toxicity case report forms.  AEs reported through expedited processes (e.g., reported to the 
IRB, FDA, etc.) must also be reported in routine study data submissions.


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8. PHARMACEUTICAL INFORMATION
A list of the adverse events and potential risks associated with the investigational agents 
administered in this study can be found in Section 7.1.
DKN-01 will be administered first followed by Nivolumab, with a minimum of 5 minutes 
between dosing.
8.1
DKN-01
8.1.1
Description
DKN-01 is an IgG4 optimized for neutralizing activity against DKK1. The molecular 
formula is C6394 H9810 N1698 O2012 S42. Molecular weight is 144,013 Daltons 
(intact).
A summary of the PK of DKN-01 after IV administration is presented below. 
Study DEK-DKK1-P100:  Summary of DKN-01 Pharmacokinetic Parameters Following IV 
Administration 
DKN-01 Dose
PK Parametera
Part A
75 mg QW
150 mg QW
300mg QW
600 mg Q2W
Part B
300 mg Q2W*
Cycle 1, Day 1
Cmax (µg/mL)
SD
N
22.8 
(7.67)
3
47.8
(4.19)
3
130
(25.7)
3
183
(34.6)
3
116
(45.0)
19
Cmax/Dose (µg/mL/mg)
SD
N
0.303
(0.102)
3
0.319
(0.0280)
3
0.434
(0.0857)
3
0.305
(0.0577)
3
0.388
(0.150)
19
AUC0–t (µgh/mL)b
SD
N
1940
(780)
3
4380
(403)
3
10900
(2320)
3
28400c
(2470)
3
17400 c*
(5430)
19
AUC0–t/Doseb(µgh/mL/mg)
SD
N
25.9
(10.4)
3
29.2
(2.68)
3
36.2
(7.73)
3
47.3
(4.11)
3
58.0
(18.1)
19
Cycle 1, Day 22
Cmax (µg/mL)
SD
N
43.2
(25.1)
3
105
(29.5)
3
224
(28.4)
3
NA
NA


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Cmax/Dose (µg/mL/mg)
SD
N
0.58576
(0.334)
3
0.701
(0.197)
3
0.748
(0.0948)
3
NA
NA
AUC0–t (µgh/mL) b
SD
N
5660
(3920)
3
15800
(5610)
3
25600
(4570)
3
NA
NA
AUC0–t/Dose (µgh/mL/mg)b
SD
N
75.5
(52.3)
3
105
(37.4)
3
85.5
(15.2)
3
NA
NA
AUC0-t = the area under the serum concentration versus time curve for the dosing interval; Cmax = peak serum concentration; 
NC = not calculated; NA = not available; SD = standard deviation
* Note that the report excluded Subject 26 (N=18) possibly because there were issues with actual times for this subject. The 
TLFs did not exclude this subject. Differences were not notable. Data from the TLFs were reported here or were used to 
recalculate the appropriate AUC values.
a Values are reported as mean (standard deviation).
b Reported as AUC0-tau in study report with tau equivalent to one week for QW dose groups vs. two weeks for Q2W dose 
groups.
c Data misreported in final TLFs, AUCs recalculated from individual values using nominal times.
8.1.2
Form
DKN-01 for Injection is supplied for clinical study use as a lyophilized formulation in a
glass vial. The drug product is composed of DKN-01 and the inactive ingredients
sucrose, polysorbate 80, sodium chloride, citric acid, and sodium citrate. 
The vial is manufactured to deliver 20 mg of DKN-01 and must be reconstituted with 
0.9% Sodium Chloride Injection, USP. The vials of lyophilized drug product do not 
contain antimicrobial preservative and must be stored in refrigerated conditions (2ºC to 
8ºC).
Reconstitution of the vial with 2.2 mL of 0.9% Sodium Chloride Injection, USP gives a 
clear solution containing 10 mg/mL DKN-01. The reconstituted vials are stable for up to 
8 hours at room temperature (up to 30°C) or refrigerated (2ºC to 8ºC). Once the 
reconstituted DKN-01 has been removed from the vial(s) into a syringe(s), it must be 
administered to the patient within 4 hours, while being stored at room temperature. 
Therefore, the maximum time limit from reconstitution to completion of administration is 
12 hours.
The route of administration is IV infusion via syringe pump only (over a period of 30 
minutes).  For patients being treated at 600mg of DKN-01, DKN-01 will be administered 
in 2 equal dose volume of appropriate sized syringes via bifurcated tubing.
8.1.3
Storage and Stability
For use in clinical studies, DKN-01 for injection is supplied as a lyophilized formulation 
in a glass vial and should be stored in refrigerated conditions (2ºC to 8ºC).


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8.1.4
Compatibility
Reconstitution of the vial with 2.2 mL of 0.9% Sodium Chloride Injection, USP gives a 
clear solution containing 10 mg/mL DKN-01. The reconstituted vials are stable for up to 
8 hours at room temperature (up to 30°C) or refrigerated (2 to 8oC). Once the 
reconstituted DKN-01 has been removed from the vial(s) into a syringe, it must be 
administered to the patient within 4 hours, while being stored at room temperature. 
Therefore, the maximum time limit from reconstitution to completion of administration is 
12 hours. 
8.1.5
Handling
Qualified personnel, familiar with procedures that minimize undue exposure to 
themselves and the environment, should undertake the preparation, handling, and safe 
disposal of the chemotherapeutic agent in a self-contained and protective environment. 
8.1.6
Availability
Drug supplies and re-supplies will be provided free of charge by Leap Therapeutics.
DKN-01 for Injection is supplied for clinical study use as a lyophilized formulation in a 
glass vial.
For more information regarding the supply of DKN-01 please refer to the study drug 
guidelines and the Pharmacy Manual (Appendix C).
8.1.7
Preparation
Reconstitution of the vial with 2.2 mL of 0.9% Sodium Chloride Injection, USP gives a 
clear solution containing 10 mg/mL DKN-01. The reconstituted vials are stable for up to 
8 hours at room temperature (up to 30°C) or refrigerated (2 to 8°C). Once the 
reconstituted DKN-01 has been removed from the vial(s) into a syringe, it must be 
administered to the patient within 4 hours, while being stored at room temperature. 
Therefore, the maximum time limit from reconstitution to completion of administration is 
12 hours. 
8.1.8
Administration
The route of administration is IV infusion via syringe pump only (over a period of 30 
minutes).  For patients being treated at 600mg of DKN-01, DKN-01 will be administered 
using a full 60ml syringe, or 2 x 30ml syringes.
8.1.9
Ordering
The investigator or designee will order DKN-01 from Leap Therapeutics according to


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ordering instructions provide by the company.  
8.1.10 Accountability
The investigator, or a responsible party designated by the investigator, should maintain a 
careful record of the inventory and disposition of the agent using the NCI Drug 
Accountability Record Form (DARF) or another comparable drug accountability form.  
(See the NCI Investigator’s Handbook for Procedures for Drug Accountability and 
Storage.)
The clinical trial centers will keep a trial specific authorization list which determines the 
persons responsible for handling of the investigational drugs. The responsible person 
regarding the following will keep accurate records in the clinical trial centers: 
•
receipt of IMP supply from sponsor (clinical trial center, principal investigator, 
identification of IMP, Batch No, Formulation, kind and size of packaging, date of 
expiry, number of study drugs per participant, number of reserve study drugs, 
number of study drugs in total, patient identification number, date and time of 
receipt); 
•
      location of storage of IMP; 
•
dispensing and returning of IMPs in the clinical trial center (date and time, 
number, batch no, patient identification number, volume of unused solution for 
injection when returning).
Accountability of the investigational study drug(s) is under the responsibility of the investigator 
and can be delegated to an appropriately qualified person. Study drug accountability should be 
maintained by each site. Accountability records should include receipt date, batch numbers, 
expiry dates, patient SeqID, use by subject, dispensing dates, quantities (lowest unit) and stock 
balance. 
8.1.11 Destruction and Return
In addition to internal accountability documentation on site, EORTC study-specific 
accountability and drug destruction forms will be supplied for this purpose, if site-specific forms 
are deemed not sufficiently detailed or do not provide enough information, according to EORTC 
Quality Assurance criteria.
The drug accountability and destruction forms will be verified during monitoring visits.
At the end of study, when all patients have stopped protocol treatment, complete drug 
reconciliation per batch should be available at the site for verification by EORTC in order to 
allow drug destruction or return procedure.
Both the unused and expired study medication must be destroyed, upon authorization of the 
sponsor, according to local regulations and procedures, and a copy of the destruction form must 
be returned to the EORTC Headquarters. 
The medication provided for this trial is to be used only as indicated in this protocol and only for 


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the patients entered in this study.
8.2
Nivolumab
8.2.1
Description
Nivolumab, also referred to as BMS-936558-01 or BMS-936558, is a soluble protein consisting
of 4 polypeptide chains, which include 2 identical heavy chains and 2 identical light chains.
Nivolumab is produced from cell culture using a Chinese Hamster Ovary (CHO) cell line. The 
molecular formula is 6362 H9862 N1712 O1995 S42. The molecular weight is 146,221 daltons.
The PK of single-agent nivolumab was studied in patients over a dose range of 0.1 to 20 
mg/kg administered as a single dose or as multiple doses of OPDIVO as a 60-minute 
intravenous infusion every 2 or 3 weeks. Nivolumab clearance (CL) decreases over time, 
with a mean maximal reduction (% coefficient of variation [CV%]) from baseline values 
of 24.5% (47.6%) resulting in a geometric mean steady-state clearance (CLss) (CV%) of 
8.2 mL/h (53.9%) in patients with metastatic tumors; the decrease in CLss is not considered 
clinically relevant. Nivolumab clearance does not decrease over time in patients with 
completely resected melanoma, as the geometric mean population clearance is 24% lower 
in this patient population compared with patients with metastatic melanoma at steady state. 
The geometric mean volume of distribution at steady state (Vss) (CV%) is 6.8 L (27.3%), 
and geometric mean elimination half-life (t1/2) is 25 days (77.5%). Steady-state 
concentrations of nivolumab were reached by 12 weeks when administered at 3 mg/kg 
every 2 weeks, and systemic accumulation was 3.7-fold. The exposure to nivolumab 
increases dose proportionally over the dose range of 0.1 to 10 mg/kg administered every 2 
weeks. The predicted exposure (Cavg and Cmax) of nivolumab after a 30-minute infusion 
is comparable to that observed with a 60-minute infusion.
Specific Populations: The population PK analysis suggested that the following factors had 
no clinically important effect on the clearance of nivolumab: age (29 to 87 years), weight 
(35 to 160 kg), gender, race, baseline LDH, PD-L1 expression, solid tumor type, tumor 
size, renal impairment, and mild hepatic impairment. 
Renal Impairment: The effect of renal impairment on the clearance of nivolumab was 
evaluated by a population PK analysis in patients with mild (eGFR 60 to 89 mL/min/1.73 
m2), moderate (eGFR 30 to 59 mL/min/1.73 m2), or severe (eGFR 15 to 29 mL/min/1.73 
m2) renal impairment. No clinically important differences in the clearance of nivolumab 
were found between patients with renal impairment and patients with normal renal 
function.
Hepatic Impairment: The effect of hepatic impairment on the clearance of nivolumab was 
evaluated by population PK analyses in patients with HCC and in patients with other 
tumors with mild hepatic impairment (total bilirubin [TB] less than or equal to the ULN 
and AST greater than ULN or TB greater than 1 to 1.5 times ULN and any AST) and in 


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HCC patients with moderate hepatic impairment (TB greater than 1.5 to 3 times ULN and 
any AST). No clinically important differences in the clearance of nivolumab were found 
between patients with mild/moderate hepatic impairment.
Full details on the clinical pharmacology aspects of nivolumab can be found in the 
Investigator Brochure.
8.2.2
Form
Nivolumab is clear to opalescent, colorless to pale yellow liquid, light (few) particulates 
may be present. The drug products are sterile, non-pyrogenic, single-use, isotonic 
aqueous solutions for intravenous (IV) infusion. Nivolumab Injection (100 mg/Vial (10 
mg/mL)) is also referred to as nivolumab injection.
8.2.3
Storage and Stability
Vials of nivolumab injection must be stored at 2°C to 8°C (36°F to 46°F) and protected 
from light and freezing. The unopened vials can be stored at room temperature (up to 
25°C, 77°F) and room light for up to 48 hours.
8.2.4
Compatibility
Care must be taken to assure sterility of the prepared solution as the product does not 
contain any antimicrobial preservative or bacteriostatic agent. Nivolumab infusions are 
compatible with polyvinyl chloride (PVC) or polyolefin containers and infusion sets, and 
glass bottles.
8.2.5
Handling
Qualified personnel, familiar with procedures that minimize undue exposure to 
themselves and the environment, should undertake the preparation, handling, and safe 
disposal of the chemotherapeutic agent in a self-contained and protective environment. 
8.2.6
Availability
Investigational product documentation must be maintained that includes all processes 
required to ensure drug is accurately administered. This includes documentation of drug 
storage, administration and, as applicable, storage temperatures, reconstitution, and use of 
required processes (eg, required diluents, administration sets).
The sites are responsible for procuring IV bags, diluent, and in-line filters.
8.2.7
Preparation
DF/HCC sites will follow institutional standard practices and/or packet insert.


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When the dose is fixed (eg, 240 mg, 360 mg, or 480 mg flat dose), nivolumab injection 
can be infused undiluted or diluted so as not to exceed a total infusion volume of 160 mL. 
For patients weighing less than 40 kilograms (kg), the total volume of infusion must not 
exceed 4 mL per kg of patient weight. During drug product preparation and handling, 
vigorous mixing or shaking is to be avoided.
The administration of nivolumab infusion must be completed within 24 hours of 
preparation. If not used immediately, the infusion solution may be stored under 
refrigeration conditions (2°C to 8°C, 36°F to 46°F) for up to 24 hours, and a maximum of 
8 hours of the total 24 hours can be at room temperature (up to 25°C, 77°F) and room 
light. The maximum of 8 hours under room temperature and room light conditions 
includes the product administration period.
8.2.8
Administration
DF/HCC sites will follow institutional standard practices and/or packet insert.
For details on prepared drug storage and administration of nivolumab, please refer to the current 
Investigator Brochure. 
Nivolumab is to be administered as a 30-minute IV infusion. At the end of the infusion, flush the 
line with a sufficient quantity of normal saline or dextrose solution.
8.2.9
Ordering
The investigator or designee will order Nivolumab from BMS according to ordering 
instructions provide by the company.  
8.2.10 Accountability
The investigator, or a responsible party designated by the investigator, should maintain a 
careful record of the inventory and disposition of the agent using the NCI Drug 
Accountability Record Form (DARF) or another comparable drug accountability form.  
(See the NCI Investigator’s Handbook for Procedures for Drug Accountability and 
Storage.)
Study drug will be administered in the clinical facility. After administration of 
nivolumab, treatment compliance will be assessed by investigator report on the case 
report forms.
8.2.11 Destruction and Return
For this study, study drugs (those supplied by BMS or sourced by the investigator) such as
partially used study drug containers, vials and syringes may be destroyed on site.


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On-site destruction is allowed provided the following minimal standards are met:

On-site disposal practices must not expose humans to risks from the drug.

On-site disposal practices and procedures are in agreement with applicable laws and 
regulations, including any special requirements for controlled or hazardous 
substances.

Written procedures for on-site disposal are available and followed. The procedures 
must be filed with the site’s SOPs and a copy provided to BMS upon request.

Records are maintained that allow for traceability of each container, including the 
date disposed of, quantity disposed, and identification of the person disposing the 
containers. The method of disposal, ie, incinerator, licensed sanitary landfill, or 
licensed waste disposal vendor must be documented.

Accountability and disposal records are complete, up-to-date, and available for the 
Monitor to review throughout the clinical trial period.
It is the investigator’s responsibility to arrange for disposal of all empty containers, provided that
procedures for proper disposal have been established according to applicable federal, state, local,
and institutional guidelines and procedures, and provided that appropriate records of disposal are
kept.
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES
9.1
Biomarker Studies
We propose baseline circulating biomarkers on all 30 patients and paired tumor biopsies in 15 
patients. Biopsies will be done at baseline and cycle 1 day 21 ± 4 days to assess early predictive 
markers of response and non-response. In responders, biopsies will be done at baseline and 
progression to assess mechanisms of resistance. The analyses will be performed in the labs of Dr. 
Dan Duda, Dr. David Ting and Dr. Nir Hacohen at the Massachusetts General Hospital Cancer 
Center.  We also propose collecting baseline and serial stools samples in patients that consent to 
this, and the analyses will be performed in Dr. Andrew Chan’s lab. 
9.1.1 Circulating biomarkers 
We will further validate blood-based immune and angiogenesis biomarkers, such as circulating 
CD4+ and CD8+ lymphocytic and myeloid-suppressor (CD14+, PD-L1+) as well as plasma 
markers, that are candidates for pharmacodynamic biomarkers or biomarkers of response to DKN-
01 (see Section 2.4B). 
Circulating tumor cells (CTCs) isolated from serial blood samples using the CTC-iChip (an 
antigen-agnostic microfluidic device) and circulating exosomal RNA will be analyzed in parallel 
by RNA-seq of the coding and non-coding transcriptomes to derive novel biomarkers. 
Additionally, ctDNA will be isolated from samples and undergo whole exome sequencing.


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9.1.2 Tumor Tissue biomarkers: 
We will evaluate baseline tumor tissue expression of the targets (DKK1 and PD-L1) to assess for 
predictors of response as well as TIL presence, tissue distribution and activation (Granzyme B, 
proliferation markers) post-therapy in any available biopsy tissue. 
We will perform single-cell RNA-sequencing and T-cell receptor (TCR) sequencing of fresh pre- 
and post-treatment biopsies to characterize the tumor microenvironment at baseline and the 
changes observed due to treatment. This data will be leveraged to identify novel predictive gene 
expression signatures, pathways and immune cell subsets relevant for response. Samples will be 
processed and analyzed by the Nir Hacohen  lab at MGH at the Center for Translational 
Immunology. In addition, the Hacohen lab will preserve core biopsies to perform spatial analysis 
by spatial transcriptomics and multiplex immunohistochemistry (IHC) to validate key findings 
by single-cell RNA-sequencing, and for RNA in situ hybridization (RNA-ISH) to detect repeat 
RNAs, which have been linked with changes in the immune microenvironment. The Hacohen  
lab has significant experience and well-validated pipelines for processing fresh tumor samples 
for single-cell RNA-sequencing and multiplex IHC, and has established pipelines for 
computational analysis of these samples. 
9.1.3 Stool Microbiome biomarkers: 
We propose to collect stool samples longitudinally throughout the study from all participants to 
understand microbial dynamics in response to treatment and identify those microbiota that may 
be used to predict disease-free progression (response), onset of treatment-related toxicities, or 
progression of disease/death. 16s and whole shotgun metagenomic/transcriptomic sequencing 
will be used to identify the changes in gut microbiota taxonomy, genomic diversity, and/or 
microbial pathways associated with drug intervention and whether there are specific associations 
that predict response, toxicity, or progression. The Chan laboratory has extensive experience in 
the handling, processing, sequencing, and data analysis required to execute these proposed 
correlative studies.
9.2
Laboratory Correlative Studies
9.2.1
Circulating Biomarkers 
Blood will be processed for CTC purification, plasma collection, and mononuclear cell isolation.  
Approximately 10 mL of blood will be processed through the CTC-iChip platform for CTC 
isolation. We will divide the CTC product into three different analyses including 1) Standard IF 
staining 2) RNA-ISH staining and 3) bulk frozen CTC product for future qRT-PCR or 
sequencing analysis.  The bulk frozen CTC product will be preserved using RNAlater 
stabilization solution (Qiagen Cat No. 76106).  RNAlater has been shown to significantly 
increase RNA recovery from CTC products and still permits the isolation and analysis of DNA 
and proteins from CTC product.  One tube of RNAlater stabilized CTC product will be purified 


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for RNA using the RNeasy Micro Kit (Qiagen Cat No. 74004). The other tube of RNAlater 
stabilized CTC product will be flash frozen and stored at -80°C for future exploratory research. 
The other 10 mL of blood will have plasma and mononuclear cells separated by Ficoll based 
density centrifugation followed by immediate flash freezing and storage at -80°C. A 1-2 mL 
aliquot of plasma will be thawed and processed for exosomes capture on the HB-ExoChip and/or 
ultracentrifugation based methods. RNA will be isolated from exosomes using TRIzol (Thermo 
Fisher Cat No. 10296028) based purification. This RNA will be either used immediately for 
analysis or stored at -80°C.
9.2.1.1
Collection of Specimen(s)
All patients will have blood drawn into four 10 mL EDTA purple top tubes for analysis before 
treatment and then every two months on treatment for the duration of the study.  
9.2.1.2
Handling of Specimens(s)
Specimens will be stored at room temperature and sent within 1 hour to the CTC laboratory for 
processing at the MGH Cancer Center, Building 149, Charlestown, MA.  
9.2.1.3
Shipping of Specimen(s)
Blood will be transported to MGH Cancer Center, Building 149, Charlestown, MA via Partners 
Healthcare approved vendor US Ground.
9.2.1.4
Site(s) Performing Correlative Study:
MGH Cancer Center – Charlestown Navy Yard Building 149
9.2.2
Tumor tissue biomarkers
Pre- and post-treatment biopsies will be collected for 1) fresh tissue, 2) fresh frozen in liquid 
nitrogen or dry ice bath, and 3) formalin for standard formalin fixed paraffin embedding (FFPE). 
We will evaluate baseline tumor tissue expression of the targets (DKK1 and PD-L1) as well as 
TIL presence, tissue distribution and activation (Granzyme B, CD69, proliferation markers) post-
therapy in any available biopsy tissue using existing antibodies against PD-L1, DKK1, 
Granzyme B, CD69 and Ki-67. The antibody used for PD-L1 will be anti-human CD274 Ab 
(clone: 29E.2A3). DKK1 will be evaluated by RNAScope. 
Fresh tissue biopsies will under single-cell RNA-sequencing using standard pipelines (10X 
genomics). Briefly, scRNAseq samples will undergo dissociation into single-cell suspension, then be 
loaded onto a Chromium Single Cell A Chip (10X Genomics) and processed through the Chromium 
Controller to generate Gel Beads in Emulsion (GEMs). RNA-seq libraries will be prepared using the 
Chromium 5’ Library and Gel Bead kit (10X Genomics) following the manufacturer’s protocol. 
Libraries will be subsequently sequenced on an Illumina HiSeqX with paired end reads. Read count 
quantification for each sample and downstream analysis will be performed according to established 


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computational pipelines and initially include unsupervised clustering and cell annotation to identify cell 
subsets, differential gene expression between selected patient and cell subsets, analysis of cellular states 
and cell-trajectories and reconstruction of TCRs to investigate T cell clonality. All analysis will be 
implemented in Seurat (v3.0) in the R programming environment.
Fresh frozen tumors will be processed for RNA and DNA extraction with the AllPrep spin 
column kit (Qiagen 80204).  RNA will be analyzed by RNA-sequencing using the Illumina Total 
RNA-seq library construction and sequencing on the NextSeq 2500 platform.  Excess RNA and 
DNA will be archived and stored at -80 degrees C.  DNA may be analyzed by whole 
genome,whole exome, or targeted gene sequencing. A single fresh frozen core will be stored for 
potential future spatial analysis with either SlideSeq or multiplex IHC. 
FFPE slides will be stained using the ACD RNAscope platform on the automated Leica Bond 
RX platform. Sequential slides will be stained for HSATII, LINE-1, TERRA, HERV-H, PPIB 
(RNA control) and other RNA markers identified by RNA-seq. 
9.2.2.1
Collection of Specimen(s)
Between one (1) and six (6) fresh, fresh frozen and FFPE biopsies will be obtained for research 
biopsies performed by interventional gastroenterology or interventional radiology.
9.2.2.2
Handling of Specimens(s)
Fresh frozen biopsies will be frozen immediately with liquid nitrogen or dry ice freezing solution 
(dry ice combined with ethanol).  FFPE samples will be placed in 10% formalin.  All samples 
will be transported immediately to the MGH Center for Translational Immunology.
9.2.2.3
Shipping of Specimen(s)
Biopsies will be transported to the MGH Center for Translational Immunology at MGH Jackson 
building on the 9th floor.
9.2.2.4
Site(s) Performing Correlative Study
MGH Center for Translational Immunology, Jackson building 9th floor.
9.2.3
Stool microbiome biomarkers
We will obtain self-collected stool specimens from participants at pre-treatment/baseline and at 
the end of each treatment cycle (returned at the time of radiologic assessment). We may 
additionally ask participants to provide a sample surrounding any treatment-related toxicities and 
a final sample at the time of progression or when the participant is taken off-treatment.
Participation in all stool collections is optional and their decision to participate in any given 
collection time point or not will not affect the participant’s participation in other aspects of the 


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trial. Therefore, we anticipate that participants may provide as few as 1 stool sample or up to 10 
stool samples. Given the individual treatment plans and the optional nature of the collection, not 
all participants will provide the same number of samples. These non-invasive stool collections 
pose no additional known risk to the participants.
We will perform RNA and DNA purification from stool aliquots and sequencing according to 
protocols optimized in our previous validation study34 in which we have collected over 932 
unique specimens and failed to recover high quality RNA and DNA from only 4 samples. 
Libraries for metagenome and metatranscriptome will be constructed using the Illumina TruSeq 
method with ~180nt inserts and sequenced on the Illumina HiSeq 2000 platform targeting a 
minimum of ~2 Gnt/sample with 100nt paired-end reads. Quality control and removal of host 
‘contaminant’ sequences will be conducted using the KneadData pipeline. Taxonomic profiles 
will be determined by MetaPhlAn2 (Metagenomic Phylogenetic Analysis), which identifies taxa 
to the species level and quantifies their relative abundances. Meta’omic data will then be piped 
into HUMAnN2 (HMP Unified Metabolic Analysis Network tools 2) to generate functional 
genomic profiles, the detailed species-specific reconstructions of microbial metabolic pathways, 
and their complement of gene orthologs, taxonomic distributions, and abundances. Within 
HUMAnN2, taxonomic specific profiles of UniRef orthologous gene families, MetaCyc, 
Unipathway, and KEGG pathways are generated to comprehensively determine the metabolic 
potential of each sample. MaAsLin, a sparse regression multivariate generalized linear model, 
will be used to test associations between clinical metadata, including trial outcomes and 
microbial community abundance and function. As this is a rapidly evolving field of study, the 
exact tools used may change, but any replacement will perform the same range of function.
9.2.3.1
Collection of Specimen(s)
Participants will be provided with an at-home stool collection kit with necessary instructions and 
will return specimens (stored in a room temperature stabilization buffer) at each clinical visit.  
Participants will be provided with a subsequent replacement kit for their next collection at each 
of these visits. 
9.2.3.2
Handling of Specimens(s)
Stool samples will be stored in a room temperature stabilization buffer provided in the at-home 
collection kits. Participants will bring samples with them to a clinical visit where study staff will 
coordinate pick-up by or delivery to a member of the Chan Laboratory. Stool samples sent to the 
Chan laboratory will be homogenized using a spatula and separated into aliquots of 100 mg for 
storage at -80°C prior to DNA and RNA extraction. All samples will be processed and frozen 
within 3 hours of receipt in the laboratory.
9.2.3.3
Shipping of Specimen(s)
Stool samples can also be shipped at room temperature (within 5 days of collection) to or 
coordinated with the laboratory team to be picked up from trial coordinators in the clinical 


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setting:
Chan Laboratory
c/o David A. Drew, Ph.D.
Massachusetts General Hospital
55 Fruit St., GRJ-815
Boston, MA 02114
Deidentified extracted DNA/RNA or stool samples may be sent to:
Broad Institute
Attn: BSP platform
301 Binney Street Lab 5076
Cambridge, MA 02142
617-714-8952
for sequencing. However, we may choose to use other comparable sequencing services according 
to resources at the time.
9.2.3.4
Site(s) Performing Correlative Study
Chan Laboratory and Broad Institute.


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STUDY CALENDAR
Baseline evaluations are to be conducted within 1 week prior to start of protocol therapy and 
laboratory parameters do not need to re-meet eligibility on c1d1 if the treating investigator feels 
it is safe and in the best interest of the patient to proceed. Scans and x-rays must be done <3 
weeks prior to the start of therapy.  
Assessments must be performed prior to administration of any study agent. Study assessments 
and agents should be administered within + 3 days of the protocol-specified date, unless 
otherwise noted.
On Treatment Period
Screening 
Cycle 1 (28 days)
Cycles 2 and 
beyond
(28 days)
Off 
Treat
ment
Pre-
Study 
(-14 days)
Day 1
Day 8
Day 15
Day 22
Day 1
Day 15
DKN-01
X
X
X
X
Nivolumab
X
X
X
X
Informed consentb
X
Demographics
X
Medical history
X
Concurrent meds
X
X-----------------------------------------------------------X
Physical exam
X
X
X
X
X
X
X
X
Vital signsc
X
X
X
X
X
X
X
X
Height
X
Weight
X
X
X
X
X
X
X
X
ECOG Performance 
status
X
X
X
X
X
X
X
X
Mismatch repair 
protein status
X
CBC w/diff
X
X
X
X
X
X
X
X
Serum chemistryd
X
X
X
X
X
X
X
X
Viral tests at 
Screening 
(HBV/HCV)e
X
Thyroid function 
testsf
X
X
X
X
Creatine 
phosphokinase 
(CPK)g
X
X
X
X
Blood coagulation 
X
X
X
X
X
X
X


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test (INR)
Amylase and lipase
X
X
X
X
X
X
X
CA19-9
X
X
X
EKG 
X
(as indicated) 
Adverse event 
evaluation
X------------------------------------------------------------X
X
Tumor 
measurements
Xh
Tumor measurements are repeated every 8 weeks.i  
X
Radiologic 
evaluation
Xh
Radiologic measurements should be performed every 
8 weeks.i
X
B-HCG
Xj
X
X
Circulating 
biomarkers (Duda 
Lab)
X
X
X
Xk
Xl
Circulating 
biomarkers (Ting 
Lab)
X
Xl
Tumor biopsym
X
X
Return at-home 
Stool Collection
X
Xn
Xn
X
a: Off-study evaluation at the time of discontinuation of treatment or up to 28 days afterwards
b: Informed consent can be signed up to 28 days prior to Cycle 1 Day 1. 
c: On treatment days, the patient’s vital signs (heart rate, respiratory rate, blood pressure, and 
temperature) should be determined within 60 minutes before and 2 hours (15 minutes) 
after the DKN-01 infusion. During infusion the vital signs should be collected as clinically 
indicated. 
d: Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, 
creatinine, glucose, LDH, phosphorus, potassium, total protein, SGOT [AST], SGPT 
[ALT], sodium.
e: Testing will be performed at Screening and will include HBV/HCV serology (HBsAg, 
HBsAb, HBcAb, and HCV antibody) and viral load assessment (HBV DNA and HCV 
RNA). Patients who have detectable HBV DNA or HCV RNA at Screening will perform 
the respective viral load test every 2 cycles (8 weeks). 
f:
TSH and free T4 will be assessed at Screening, Baseline, and every 2 cycles thereafter (8 
weeks). T3 or free T3 levels will be assessed at Screening and Baseline. Patient can receive 
dose prior to receiving the results for thyroid labs.
g: Patient can receive dose prior to receiving the results for CPK.
h: Radiologic evaluation within 21 days of c1d1
i:
The window for tumor measurements and radiologic evaluation is +/- 7 days
j:
Serum pregnancy test (women of childbearing potential) at baseline and every 4 weeks.  
For day 1 of each cycle (including c1d1), serum pregnancy test needs to be within 24 hours 
of nivolumab dosing.
k: Circulating biomarkers will be collected on c2d1 but not on day 1 of subsequent cycles.  
See Biomarker Cheat Sheet on Alert page, section 9, and Appendix B for full details.
l:
Circulating biomarkers should be collected at progression


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m: Fifteen patients will have paired tumor biopsies while on treatment.  Biopsies will be done 
at baseline (within 28 days of c1d1) and at c1d22 (+/- 4 days) to assess early predictive 
markers of response and non-response. In 4 responders, biopsies will be done at baseline 
and progression to assess mechanisms of resistance.  If the treating investigator feels the 
biopsy is unsafe or not feasible for the patient, they may reach out to the Principle 
Investigator for discussion of omission of biopsy.
n: Participants who consent to optional stool collection will collect samples at home prior to 
the start of each treatment cycle but return these on the first day of each treatment cycle. 
Patients will ideally provide a stool sample every cycle, but the number of samples they 
provide over time is voluntary
o:
10. MEASUREMENT OF EFFECT
Objective tumor response and time to progression will be measured in this study using the 
revised international criteria (1.1) proposed by the RECIST (Response Evaluation Criteria in 
Solid Tumors) committee. 
The complete RECIST criteria are included in the published RECIST document available at 
http://www.eortc.org/RECIST.
10.1
Antitumor Effect – Solid Tumors
For the purposes of this study, participants should be re-evaluated for response every 8 weeks.  
In addition to a baseline scan, confirmatory scans should also be obtained usually at the next 
schedule scan vist but not less than 4 weeks following initial documentation of objective 
response.
Response and progression will be evaluated in this study using the new international criteria 
proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1) 
[Eur J Ca 45:228-247, 2009].  Changes in the largest diameter (unidimensional measurement) of 
the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the 
RECIST criteria.
10.1.1 Definitions
Evaluable for Target Disease response.  Only those participants who have measurable 
disease present at baseline, have received at least one cycle of therapy, and have had their 
disease re-evaluated will be considered evaluable for target disease response.  These 
participants will have their response classified according to the definitions stated below.  
(Note:  Participants who exhibit objective disease progression prior to the end of cycle 1 
will also be considered evaluable.)
Evaluable Non-Target Disease Response.  Participants who have lesions present at 
baseline that are evaluable but do not meet the definitions of measurable disease, have 
received at least one cycle of therapy, and have had their disease re-evaluated will be 


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


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disease. 
Non-target lesions.  All other lesions (or sites of disease) including any measurable 
lesions over and above the 5 target lesions should be identified as non-target lesions and 
should also be recorded at baseline. Measurements of these lesions are not required, but 
the presence, absence, or in rare cases unequivocal progression of each should be noted 
throughout follow up. 
10.1.3 Methods for Evaluation of Disease
All measurements should be taken and recorded in metric notation using a ruler, calipers, 
or a digital measurement tool.  All baseline evaluations should be performed as closely as 
possible to the beginning of treatment and never more than 4 weeks before the beginning 
of the treatment.
The same method of assessment and the same technique should be used to characterize 
each identified and reported lesion at baseline and during follow-up. Imaging-based 
evaluation is preferred to evaluation by clinical examination unless the lesion(s) being 
followed cannot be imaged but are assessable by clinical exam. 
Clinical lesions. Clinical lesions will only be considered measurable when they are 
superficial (e.g., skin nodules and palpable lymph nodes) and ≥10 mm in diameter as 
assessed using calipers (e.g., skin nodules).  In the case of skin lesions, documentation by 
color photography, including a ruler to estimate the size of the lesion, is recommended. 
Chest x-ray.  Lesions on chest x-ray are acceptable as measurable lesions when they are 
clearly defined and surrounded by aerated lung; however, CT is preferable. 
Conventional CT and MRI. This guideline has defined measurability of lesions on CT 
scan based on the assumption that CT thickness is 5mm or less. If CT scans have slice 
thickness greater than 5 mm, the minimum size of a measurable lesion should be twice 
the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans).  
Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which 
greatly impact image quality, lesion conspicuity, and measurement.  Furthermore, the 
availability of MRI is variable globally.  As with CT, if an MRI is performed, the 
technical specifications of the scanning sequences used should be optimized for the 
evaluation of the type and site of disease.  Furthermore, as with CT, the modality used at 
follow-up should be the same as was used at baseline and the lesions should be 
measured/assessed on the same pulse sequence.  It is beyond the scope of the RECIST 
guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body 
parts, and diseases.  Ideally, the same type of scanner should be used and the image 
acquisition protocol should be followed as closely as possible to prior scans.  Body scans 
should be performed with breath-hold scanning techniques, if possible.


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FDG-PET. While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease).  New lesions on the basis 
of FDG-PET imaging can be identified according to the following algorithm: 
(a) Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of 
PD based on a new lesion.
(b)  No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive 
FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, this is 
PD.  If the positive FDG-PET at follow-up is not confirmed as a new site of disease 
on CT, additional follow-up CT  scans are needed to determine if there is truly 
progression occurring at that site (if so, the date of PD will be the date of the initial 
abnormal FDG-PET scan).  If the positive FDG-PET at follow-up corresponds to a 
pre-existing site of disease on CT that is not progressing on the basis of the anatomic 
images, this is not PD.
(c)  FDG-PET may be used to upgrade a response to a CR in a manner similar to a 
biopsy in cases where a residual radiographic abnormality is thought to represent 
fibrosis or scarring.  The use of FDG-PET in this circumstance should be 
prospectively described in the protocol and supported by disease-specific medical 
literature for the indication.  However, it must be acknowledged that both approaches 
may lead to false positive CR due to limitations of FDG-PET and biopsy 
resolution/sensitivity.
Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake 
greater than twice that of the surrounding tissue on the attenuation corrected image.
10.1.4 Response Criteria
10.1.4.1
Evaluation of Target Lesions
Complete Response (CR):  Disappearance of all target lesions.  Any pathological 
lymph nodes (whether target or non-target) must have reduction in short axis to <10 
mm.
Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target 
lesions, taking as reference the baseline sum diameters.
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of 
target lesions, taking as reference the smallest sum on study (this includes the 
baseline sum if that is the smallest on study).  In addition to the relative increase of 
20%, the sum must also demonstrate an absolute increase of at least 5 mm.  (Note:  
the appearance of one or more new lesions is also considered progressions).
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum diameters while on 


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study.
10.1.4.2
Evaluation of Non-Target Lesions
Complete Response (CR):  Disappearance of all non-target lesions and normalization 
of tumor marker level.  All lymph nodes must be non-pathological in size (<10 mm 
short axis).
Note:  If tumor markers are initially above the upper normal limit, they must 
normalize for a patient to be considered in complete clinical response.
Non-CR/Non-PD:  Persistence of one or more non-target lesion(s) and/or 
maintenance of tumor marker level above the normal limits.
Progressive Disease (PD):  Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target lesions.  Unequivocal progression 
should not normally trump target lesion status.  It must be representative of overall 
disease status change, not a single lesion increase.    
Although a clear progression of “non-target” lesions only is exceptional, the opinion 
of the treating physician should prevail in such circumstances, and the progression 
status should be confirmed at a later time by the review panel (or Principal 
Investigator).
10.1.4.3
Evaluation of New Lesions
The finding of a new lesion should be unequivocal (i.e. not due to difference in 
scanning technique, imaging modality, or findings thought to represent something 
other than tumor (for example, some ‘new’ bone lesions may be simply healing or 
flare of pre-existing lesions). However, a lesion identified on a follow-up scan in an 
anatomical location that was not scanned at baseline is considered new and will 
indicate PD. If a new lesion is equivocal (because of small size etc.), follow-up 
evaluation will clarify if it truly represents new disease and if PD is confirmed, 
progression should be declared using the date of the initial scan on which the lesion 
was discovered.
10.1.4.4
Evaluation of Best Overall Response
The best overall response is the best response recorded from the start of the treatment 
until disease progression/recurrence (taking as reference for progressive disease the 
smallest measurements recorded since the treatment started).  The patient's best 
response assignment will depend on the achievement of both measurement and 
confirmation criteria.
Response (partial or complete) according to RECIST 1.1 with confirmation requires a 
confirmatory scan preferably at the next regularly scheduled imaging visit and no 


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earlier than 4 weeks after the prior assessment of response. 
For Participants with Measurable Disease (i.e., Target Disease)
Target 
Lesions
Non-Target 
Lesions
New 
Lesions
Overall 
Response
Best Overall Response when 
Confirmation is Required*
CR
CR
No
CR
>4 wks Confirmation
CR
Non-CR/Non-
PD
No
PR
CR
Not evaluated
No
PR
PR
Non-CR/Non-
PD/not 
evaluated
No
PR
>4 wks Confirmation
SD
Non-CR/Non-
PD/not 
evaluated
No
SD
Documented at least once >4 
wks from baseline
PD
Any
Yes or No
PD
Any
PD**
Yes or No
PD
Any
Any
Yes
PD
no prior SD, PR or CR

See RECIST 1.1 manuscript for further details on what is evidence of a new lesion.
**      In exceptional circumstances, unequivocal progression in non-target lesions may be
            accepted as disease progression.
Note:  Participants with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be 
reported as “symptomatic deterioration.”  Every effort should be made to document the 
objective progression even after discontinuation of treatment.
For Participants with Non-Measurable Disease (i.e., Non-Target Disease)
Non-Target Lesions
New Lesions
Overall Response
CR
No
CR
Non-CR/non-PD
No
Non-CR/non-PD*
Not all evaluated
No
not evaluated
Unequivocal PD
Yes or No
PD
Any
Yes
PD

‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since SD is 
increasingly used as an endpoint for assessment of efficacy in some trials so to assign 
this category when no lesions can be measured is not advised
10.1.5 Duration of Response
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first 
date that recurrent or progressive disease is objectively documented (taking as reference 


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for progressive disease the smallest measurements recorded since the treatment started, or 
death due to any cause. Participants without events reported are censored at the last 
disease evaluation).
Duration of overall complete response: The duration of overall CR is measured from the 
time measurement criteria are first met for CR until the first date that progressive disease 
is objectively documented, or death due to any cause. Participants without events 
reported are censored at the last disease evaluation.
Duration of stable disease:  Stable disease is measured from the start of the treatment 
until the criteria for progression are met, taking as reference the smallest measurements 
recorded since the treatment started, including the baseline measurements. 
10.1.6 Progression-Free Survival
Overall Survival: Overall Survival (OS) is defined as the time from randomization (or 
registration) to death due to any cause, or censored at date last known alive.
Progression-Free Survival: Progression-Free Survival (PFS) is defined as the time from 
randomization (or registration) to the earlier of progression or death due to any cause. 
Participants alive without disease progression are censored at date of last disease 
evaluation.
Time to Progression: Time to Progression (TTP) is defined as the time from 
randomization (or registration) to progression, or censored at date of last disease 
evaluation for those without progression reported.
10.1.7 Response Review
Radiological images will be independently reviewed by experts independent of the study 
in our Tumor Imaging Metrics Core (TIMC). 
11. DATA REPORTING / REGULATORY REQUIREMENTS
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements).
11.1
Data Reporting
11.1.1 Method
The Office of Data Quality (ODQ) will collect, manage, and perform quality checks on 
the data for this study.


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11.1.2 Responsibility for Data Submission
Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or 
data forms to the Office of Data Quality (ODQ) in accordance with DF/HCC policies.
11.2
Data Safety Monitoring
The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor toxicity 
and accrual data from this study. The committee is composed of medical oncologists, research 
nurses, pharmacists and biostatisticians with direct experience in cancer clinical research.  
Information that raises any questions about participant safety will be addressed with the Overall 
PI and study team.
The DSMC will review each protocol up to four times a year with the frequency determined by 
the outcome of previous reviews. Information to be provided to the committee may include: up-
to-date participant accrual; current dose level information; DLT information; all grade 2 or 
higher unexpected adverse events that have been reported; summary of all deaths occurring 
within 30 days of intervention for Phase I or II protocols; for gene therapy protocols, summary of 
all deaths while being treated and during active follow-up; any response information; audit 
results, and a summary provided by the study team. Other information (e.g. scans, laboratory 
values) will be provided upon request. 
11.3
Collaborative Agreements Language N/A
This is an investigatory initiated study sponsored by Leap Therapeutics and BMS. Data will be 
shared between the participating institution and the two companies 
12. STATISTICAL CONSIDERATIONS
12.1
Study Design/Endpoints
The primary endpoint is to investigate the overall response rate (ORR) of the combination of DKN-
01 and nivolumab. 
We hypothesize that the combination of DKN-01 and nivolumab will have a true ORR  20%, while 
the null hypothesis would be < 5%.  The ORR of 5% was selected on the basis of studies of 
systemic therapy beyond the first line in unselected patients with refractory advanced biliary tract 
cancer showing an ORR in the single digits35.   
To detect a difference of 15% (20% vs 5%) with 80% power and using a one-sided binomial test 
with 5% type 1 error, we require a total of 27 patients. We recommend enrolling 30 patients to 
allow for 10% attrition. The anticipated follow-up is 12 months. 


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We will use a Simon’s two-stage design. After testing the treatment on 13 patients in the first 
stage, the trial will be terminated if 0 respond. If the trial goes on to the second stage, a total of 
27 patients will be studied. If the total number responding is less than or equal to 3, the treatment 
is rejected. If the chosen dose is 300mg, the patients treated at 600mg will not be included in the 
two-stage design. The patients treated at 600mg will not be replaced.
If the treatment is actually not effective, there is a 0.042 probability of concluding that it is. If the 
treatment is actually effective, there is a 0.199 probability of concluding that it is not. 
All patients with measurable disease who receive at least one dose of both DKN-01 and 
nivolumab will be considered evaluable.
Secondary Endpoints:
1) Efficacy Endpoints:

Progression Free Survival (PFS) – PFS is defined using the RECIST criteria for progressive 
disease. Patients discontinued from study for clinical progression will also be evaluable for 
this endpoint.  

Median Overall Survival (OS) – Median OS is defined as the length of time from trial 
registration that half of the enrolled patients in the study are still alive.  Kaplan-Meier estimates 
of OS rates will be calculated along with their corresponding 95% confidence intervals. Cox 
proportional hazards regression modeling of OS will be used to assess the effect of the 
combination of DKN-01 and nivolumab on OS while controlling for other confounders.

ORR, PFS and OS of subgroups stratified by tumor DKK1 and PD-L1 expression status in a 
pre-planned post-hoc analysis will also be assessed.
2) Biologic correlative studies of target inhibition – Categorical measures related to each marker 
will be correlated with response to the combination of DKN-01 and nivolumab using Fisher’s exact 
tests. Kaplan-Meier survival curves and Cox proportional hazard models will be used to explore 
the relationship between the biomarker categories and PFS and OS as described in detail in section 
9.4. These correlative endpoints are exploratory in nature. 
3) Tolerability and safety – All enrolled patients who receive at least one dose of DKN-01 or 
nivolumab will be assessed for adverse events by the Common Toxicity Criteria for Adverse 
Events, v4.03, and the relationship of each AE to the study drugs will be documented. 
12.2
Sample Size, Accrual Rate and Study Duration
A total of 30 evaluable participants will be accrued within 24 months. The anticipated follow-up 
is 12 months. The total planned study duration is 36 months.
Accrual Targets
Sex/Gender
Ethnic Category
Females
Males
Total
Hispanic or Latino
2
+
1
=
3


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Not Hispanic or Latino
15
+
12
=
27
Ethnic Category: Total of all 
subjects
17
+
13
=
30
Racial Category
American Indian or Alaskan 
Native
0
+
0
=
0
Asian
2
+
2
=
4
Black or African American
1
+
1
=
2
Native Hawaiian or other Pacific 
Islander
0
+
0
=
0
White
14
+
10
=
30
Racial Category: Total of all 
subjects
17
+
13
=
30
12.3
Stratification Factors
The patients will be stratified in a pre-planned post-hoc analysis by PD-L1status and DKK1 
status.
12.4
Interim Monitoring Plan
An early stopping rule is planned, which states that if the ORR is 0% after 13 patients are treated, 
we will terminate the study.  
12.5
Analysis of Primary Endpoints
See Section 12.1.
12.6
Analysis of Secondary Endpoints
See Section 12.1.
12.7
Reporting and Exclusions
12.7.1 Evaluation of Toxicity
All patients who receive at least 1 dose of at least one study drug will be considered 
evaluable for toxicity.
12.7.2 Evaluation of the Primary Efficacy Endpoint


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Total accrual may be adjusted to account for patients inevaluable for response, ie those patients 
not receiving at least one dose of the study drugs. Participants that are registered for the trial but 
do not begin treatment will be replaced and not included in the analysis of the primary endpoint.
13. PUBLICATION PLAN
The results should be made public within 24 months of reaching the end of the study. The end of 
the study is the time point at which the last data items are to be reported, or after the outcome 
data are sufficiently mature for analysis, as defined in the section on Sample Size, Accrual Rate 
and Study Duration. If a report is planned to be published in a peer-reviewed journal, then that 
initial release may be an abstract that meets the requirements of the International Committee of 
Medical Journal Editors. A full report of the outcomes should be made public no later than three 
(3) years after the end of the study. 
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APPENDIX A
PERFORMANCE STATUS CRITERIA
ECOG Performance Status Scale
Grade
Descriptions
0
Normal activity.  Fully active, able to carry on all pre-disease performance without 
restriction.
1
Symptoms, but ambulatory.  Restricted in physically strenuous activity, but ambulatory and 
able to carry out work of a light or sedentary nature (e.g., light housework, office work).
2
In bed <50% of the time.  Ambulatory and capable of all self-care, but unable to carry out 
any work activities.  Up and about more than 50% of waking hours.
3
In bed >50% of the time.  Capable of only limited self-care, confined to bed or chair more 
than 50% of waking hours.
4
100% bedridden.  Completely disabled.  Cannot carry on any self-care.  Totally confined to 
bed or chair.
5
Dead.


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APPENDIX B BIOASSAY TEMPLATES
Duda lab
Test and Control Articles
Material
Source
Human ProInflammatory Panel 1 V-PLEX™ Plus 
(Cat #K15049G-2)
Meso-Scale Discovery
Human Angiogenesis Panel 1 V-PLEX Kit (Cat 
#K15190D-2):
Meso-Scale Discovery
Human CXCL12/SDF1alpha Quantikine ELISA Kit 
(Cat #DSA00)
R&D Systems
Human HGF Quantikine ELISA Kit (Cat #DHG00)
R&D Systems
Plasma and peripheral blood mononuclear cells will be collected from all patients with advanced 
carcinoma enrolled in this clinical trial. We will measure potential biomarkers at baseline (pre-
treatment) and then weekly for the first cycle of treatment (Days 8, 15), pre-treatment on Cycle 2 
Day 1 and at the end of treatment (or early termination). 
Experimental Methods
Eight cc of blood were collected in purple top (plasma EDTA) vacutainers, with a minimum of 
5cc required. All samples were shipped on dry ice to the Steele Laboratories at MGH. Blood was 
separated by centrifugation in refrigerated centrifuge for 20 min at 2000 rpm into plasma and a 
cellular phase. The plasma was prepared in standard fashion and stored at –78 degrees Celsius 
until analysis. Plasma analysis was carried out for a panel of circulating inflammatory and 
angiogenic molecules previously identified as potential biomarkers. All measurements were 
performed in duplicate in a CLIA-Certified Core. 
1.
Human ProInflammatory Panel: IFN-γ, IL-1β, IL-10, IL-12 p70, IL-13, IL-2, IL-4,
IL-6, IL-8, TNF-α
2.
Human Angiogenesis Panel: bFGF, sFLT-1/sVEGFR1, PlGF, sTIE-2, VEGF, VEGF-
C, VEGF-D
3.
Human SDF1alpha and HGF 
Each participant will have 2 vials of blood (~8 mL each) collected at the time points listed in 
Section 2.1. The blood must be processed and stored within 30-45 minutes after collection. 
Materials and Labeling of Blood Tubes and Cryovials
Labels will be provided for the vacutainer blood tubes and the cryovials. All labels will have 
space for sites to provide the following information:

Participant study number;

Participant initials and date of birth


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
Time point when sample was collected;

For the blood tubes, a “BL” and for the plasma cryovials, a “PL” to distinguish which 
label should be used for each tube.
The site will be responsible for obtaining the appropriate tubes for the blood and plasma 
specimens. The list of supply options are as follows:
For Blood Draw Tubes (3 Options)
1.
SARSTEDT Monovette® EDTA KE (9 mL), Part # 02.1333.001; or 
2.
Becton-Dickinson VacutainerTM K2E (10 mL), Part # 367525; or 
3.
Greiner Bio-One Vacuette® K3E EDTA K3 (9 mL), Part # 455036; and
For Plasma Cryovials (1 Option)
4.
Nunc, Internally Threaded Cryotube, (3.6 mL vials), Fischer Scientific, Part # 12-
565-162N.
Blood Draw and Centrifugation

Prior to the blood draw, the blood vacutainer should be pre-cooled by placing tube in a 
bucket with wet ice.

Directly from the participant’s IV line, draw the 2 vials of blood using 2 pre-labeled 
blood vacutainer tubes. 

Record time of blood draw on Blood Processing Form (BPF).

Immediately invert the blood tube several times to ensure proper mixing with the 
preservative.

Place the blood tube in a bucket filled with wet ice.

Centrifuge the blood tube at 700g (2000rpm) for 20 minutes at 4°C with no break at the 
end of centrifugation for plasma extraction.

Using a sterile pipette, pipette the top clear layer (careful not to disturb the bottom red 
layer) and aliquot equally into 3 pre-labeled 1.8 mL Nunc cryovials.

Visually check the plasma for hemolysis. 

If the plasma appears to be yellow and clear, please proceed with processing the plasma 
and record the observation on the BPF form. 

If the plasma appears to be dark red, please discard the plasma and sign and date the BPF.

After the plasma has been extracted, check the red blood cells by sticking two wooden 
applicator sticks in the tube and observe the sticks for clots. Record if there was clotting 
observed, check yes or no on the BPF.

Record numbers of vials on BPF.

Immediately store the cryovials into a –80°C freezer*.

Record time of freeze, location of the vials (i.e., freezer number, shelf, box number, and 
room #, as applicable) on the BPF.
* 
If a –80°C freezer is not available on site, the plasma specimens should be shipped to 
the core facility on dry ice the same day as processing. See Section 4.0 for shipping procedures.
Labeling and Storing Specimens 
For tracking purposes, each specimen and its associated forms should be labeled with the site 


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number and case number. All cryovials should be stored in a monitored –80° C freezer.  A 
separate freezer box should be set a side for the storage of all the plasma vials. They will be 
shipped in bulk to the testing core facility at completion of study.
SHIPPING  SPECIMENS TO CORE FACILITY
When blood collection has been completed for all participants and after the participants go off 
trial the plasma samples should be shipped to the Steele Laboratory at Massachusetts General 
Hospital on dry ice in a Styrofoam box. The Styrofoam container should be packed with at least 
7 pounds of dry ice, and make sure the top is completely covered with dry ice.  Seal the 
Styrofoam container within a tight-fitting cardboard shipping box.  A copy of the Plasma 
Shipping (PS) Form for each set of samples should be placed within a separate zip-lock plastic 
bag and placed on top of the Styrofoam container lid before the external shipping box is sealed.
If the site does not have –80°C storage capability, the plasma samples should be sent to the 
Steele Laboratory at Massachusetts General Hospital after the blood has been processed. The 
Styrofoam container should be packed with at least 2 inches of dry ice on the bottom, and 
completely covered on the top with an additional 2 inches or more of dry ice. Seal the Styrofoam 
container within a tight-fitting cardboard shipping box.  A copy of the Plasma Shipping (PS) 
Form for each set of samples should be placed within a separate zip-lock plastic bag and placed 
on top of the Styrofoam container lid before the external shipping box is sealed.
If the site does not have facilities and trained personnel for plasma separation, the blood samples 
should be shipped to the Steele Laboratory at Massachusetts General Hospital with cold packs in 
a Styrofoam box within 24 hours (DO NOT FREEZE).  Seal the Styrofoam container within a 
tight-fitting cardboard shipping box.  A copy of the Plasma Shipping (PS) Form for each set of 
samples should be placed within a separate zip-lock plastic bag and placed on top of the 
Styrofoam container lid before the external shipping box is sealed.
Specimens should be shipped Monday to Wednesday only by overnight FedEx to the Testing 
Core Facility. On the day of shipment, the study coordinator will notify the Testing Core Facility 
via e-mail at annak@steele.mgh.harvard.edu or FAX (617-724-5841, ATTN: A. Khachatryan or 
J. Kahn) so they know to expect the upcoming shipment.  Include the estimated date of arrival 
and the FedEx tracking number.  
NOTE:  The subject line of the e-mail/FAX should include the following so that the Testing Core 
Facility staff can distinguish plasma specimens sent by the sites. 
Plasma Specimen Shipment--Site Name
Upon receipt of specimens, the Testing Core Facility will reconcile the materials and notify the 
site study coordinator of missing specimens, damaged specimens, or any concerns to be 
addressed. 
Shipping Materials and Process
The appropriate shipping materials for specimens are the following:

Storage boxes for plasma tubes (Fisherbrand, 5 3/4” x 5 3/4” x 4 7/8”, Part # 03-395-01, 
and dividers, Part # 03-395-11);

Large size zip-locked bags;


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
Multi-purpose insulated bio-shippers (Thermosafe Bio-Shippers; dimensions 14” x 10” x 
14”); 

Biohazard bags;

M3 carton sealing tape;

Shipping labels to indicate: “Notice: Keep Frozen” use only for Dry Ice shipments, 
Upright arrows,  “Diagnostic Specimens – Not restricted, Packed in Compliance with 
IATA Packing Instruction 650”, and “Class 9 – Dry Ice” label; and “Keep Refrigerated” 
use only for Cold Pack shipments.

Dry ice or Cold Packs
The packing process for dry ice shipments includes the following: 

Place all plasma tubes in storage freezer boxes, tape the sides of the boxes; 

Place one freezer box, each separately, in a large zip-locked bag;

Pack the Styrofoam container with at least 7 pounds of dry ice;

Place the bagged freezer boxes in the middle of the bio-shipper (you can fit as many as 
the box allows);

Pack the Styrofoam container with an additional dry-ice on the top of the boxes to cover 
the top;  

Place a copy of the Plasma Shipping Form (PS) for a single participant inside one 
biohazard bag, in the form slot (use as many forms/bags as necessary to cover the 
contents of the box to be shipped); 

Close the lid, place all bagged shipping forms on top of the lid, and seal the shipping 
container with tape.

Maintain a copy of the transmittal log at the site.

The packing process for cold pack shipments includes the following: 

Place all blood tubes in storage freezer boxes, tape the sides of the boxes

Place one freezer box, each separately, in a large zip-locked bag.

Place the bagged freezer boxes in the middle of the bio-shipper (you can fit as many as 
the box allows)

Pack the Styrofoam container with 4-6 cold packs surrounding the boxes with the blood 
tubes.

Place a copy of the Plasma Shipping Form (PS) for a single participant inside one 
biohazard bag, in the form slot (use as many forms/bags as necessary to cover the 
contents of the box to be shipped).

Close the lid, place all bagged shipping forms on top of the lid, and seal the shipping 
container with tape.

Maintain a copy of the transmittal log at the site.
Labeling Shipping Containers
Label each shipping container with the FedEx shipping label to include the following:
1.
The study coordinator’s return address.
2.
The Testing Core Facility address:
ATTN: Mrs. Anna Khachatryan / Mrs. Julia Kahn


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MGH, Cox-734
100 Blossom St.
Boston, MA 02114, USA
Phone: (617) 726-4088 or (617) 726-8143 or (617) 724-1353
Fax: (617) 724-5841
Pager: 14082
Email: annak@steele.mgh.harvard.edu, julia@steele.mgh.harvard.edu
3.
“Notice: Keep Frozen”, “Class 9 – Dry Ice” stickers or “Keep Refrigerated”, Upright 
arrows, and “Diagnostic Specimens – Not restricted, Packed in Compliance with IATA Packing 
Instruction 650”. 
Summary Shipping Task List
The following summarizes the tasks to be completed by the site for a scheduled shipment:

Prepare transmittal paperwork and retain copies at the site.

Send a notification e-mail or FAX to the Testing Core Facility listing the items being 
shipped, including:  the FedEx tracking number, total number of plasma tubes in the 
shipment, and the expected date of arrival. Please note “Specimen Shipment—Site 
Name.” in the e-mail or FAX ‘Subject’ line. 

Pack the plasma specimens according to instructions above. 

Label each shipping container with the FedEx shipping label and affix all appropriate 
shipping labels.

Maintain a copy of the transmittal log at the site.
Cellular Biomarkers
Circulating mononuclear cells will be collected for analysis of lymphocytic and myeloid 
populations.
Collection and Handling Procedures for Cellular biomarkers for Dr. Duda’s lab (Steele 
Laboratories)
•
Collect blood in an 8.0 ml Sodium Citrate tube (Blue Tiger Top Tube, see 
http://www.bd.com/vacutainer/products/molecular/citrate/)
•
Invert several times to ensure mixing with the anticoagulant.
•
Place a label on this blue tiger top vial of blood and cover the label with freezer tape.
•
Place the labeled vial on wet ice and send to lab for further processing.
Processing of Specimen 
•
Within two hours of collection of the blue tiger top tube, the lab will centrifuge the 
blue tiger top tube at 4ºC temperature in a horizontal rotator for 25 minutes at 1600 g.
•
After centrifugation, the mononuclear cells will be visible in a whitish layer just 
under the plasma. Carefully remove (and discard) the top plasma layer. Transfer the 
mononuclear layer (500-900 microliters) in the CPT tube to a labeled cyrovial. (Nunc. Product 
#377267)


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•
Add equal volume of freezing media (RPMI-1640 media with 20% dimethyl 
sulfoxide is then added. The cryotubes should be labeled and immediately be placed in a -80ºC 
degree freezer.
Sample Labels for Lab Samples 
Complete the labels with patient identifiers printing each label with Study-No., patient ID, 
initials and day/time of sample collection (24-hour clock format, i.e., 6:30 pm = 18:30). A label 
example is provided below:
Study-No.:Investigator:
Patient-ID:Patient Initials: 
Date of birth:
Date of sampling: (mm/dd/yy)
Time of Sampling: (hh:mm) (24-hr)
Sample Type: (Cells or Plasma)


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APPENDIX C NEW YORK HEART ASSOCIATION [NYHA] CLASSIFICATION 
Class
Patient Symptoms
I
No limitation of physical activity. Ordinary physical activity does not cause undue 
fatigue, palpitation, dyspnea (shortness of breath).
II
Slight limitation of physical activity. Comfortable at rest. Ordinary physical activity 
results in fatigue, palpitation, dyspnea (shortness of breath).
III
Marked limitation of physical activity. Comfortable at rest. Less than ordinary 
activity causes fatigue, palpitation, or dyspnea.
IV
Unable to carry on any physical activity without discomfort. Symptoms of heart 
failure at rest. If any physical activity is undertaken, discomfort increases.