Conditions: Melanoma Stage Iii Melanoma Stage Iv Melanoma Melanoma (Skin) Cutaneous Melanoma
Keywords: Denosumab Pembrolizumab Melanoma Unresectable Cutaneous Nivolumab
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 25
Healthy volunteers: no

Brief summary:
This is a multicenter open-label, single-arm, phase II study designed to investigate the pharmacodynamic and antitumor effects of denosumab alone and in combination with an anti-Programmed death-1 or Programmed death ligand 1 (PD1) agent (pembrolizumab or nivolumab) in patients with unresectable Programmed death-1 or Programmed death ligand 1 (PD-1/PD-L1) inhibitor-naïve regional and distant metastatic melanoma (The American Joint Committee on Cancer (AJCC) stage III/IV). The pharmacodynamic and antitumor effects will be investigated by performing translational research on peripheral blood and tumor tissue collected before and during denosumab alone and in combination with anti-PD-1 treatment.

Detailed description:
STUDY OBJECTIVES

Co-primary Objectives

* Assess the mechanistic (immune-mediated and/or direct antitumor effect) and pharmacodynamics effect (tissue saturation studies) of denosumab alone (i.e., after three loading doses of denosumab are given on days 1,8 and 22) in patients with unresectable (or resectable) stage III or distant metastatic PD-1/PD-L1 inhibitor-naïve cutaneous melanoma (stage III/IV) by performing translational research on peripheral blood and tumor biopsy samples collected at baseline and after third loading dose of Denosumab.
* Assess the immune-mediated and direct antitumor effect of denosumab in combination with anti- PD-1 agent in patients with unresectable (or resectable) stage III or distant metastatic PD-1/PD-L1 inhibitor-naïve cutaneous melanoma (The American Joint Committee on Cancer (AJCC) stage III/IV) by performing translational research on peripheral blood and tumor biopsy samples collected at weeks 16, 28 and 40 of the study and comparing the results with those from baseline and after third loading dose of Denosumab.

Secondary Objectives

* Assess the safety of the denosumab-anti-PD-1 agent combination in unresectable (resectable) stage III or distant metastatic PD-1/PD-L1 inhibitor-naïve melanoma (The American Joint Committee on Cancer (AJCC) stage III/IV) by National Cancer Institute (NCI)-Common Terminology Criteria for Adverse Events (CTCAE) v.5.0.
* Determine antitumor response by Response evaluation criteria in solid tumors (RECIST) v1.1 criteria of the denosumab-anti- PD-1 agent combination at 16 weeks in patients with unresectable (resectable) stage III or distant metastatic PD-1/PD-L1 inhibitor-naïve melanoma (The American Joint Committee on Cancer (AJCC) stage III/IV).
* Determine the 1-year Overall Survival rate of the Denosumab-anti-PD-1 agent combination in patients with unresectable (or resectable) stage III or distant metastatic PD-1/PD-L1 inhibitor-naïve melanoma (The American Joint Committee on Cancer (AJCC) stage III/IV).
* Determine the 6-month PFS rate of the denosumab-anti-PD-1 agent combination in patients with unresectable (or resectable) stage III or distant metastatic PD-1/PD-L1 inhibitor-naive melanoma (The American Joint Committee on Cancer (AJCC) stage III/IV).

Endpoints Co-primary Endpoints

* The immune-mediated mechanism of action of denosumab alone will be evaluated in blood and tumor samples collected at baseline and after the third loading dose of denosumab. Multiparameter flow cytometry and ELISA assays will be performed on peripheral blood/serum samples as outlinedin the protocol. Tumor biopsy samples will be evaluated by IHC and IF studies as outlined in the protocol.
* The immune-mediated mechanism of action of denosumab combined with anti-PD-1 agent will be evaluated in blood and tumor samples collected at weeks 16, 28, and 40 of the study. Multi-parameter flow cytometry and ELISA assays will be performed on peripheral blood/serum samples collected at weeks 16, 28, and 40 as outlined in the protocol. Tumor biopsy samples obtained at week 16 will be evaluated by immune histochemical (IHC) and immune florescent (IF) studies as outlined in the protocol.

Secondary Endpoints

* Adverse Events (AE) experienced by patients receiving denosumab-anti- PD-1 agent will be assessed per National Cancer Institute (NCI)-Common Terminology Criteria for Adverse Events (CTCAE) v.5.0.
* The overall response (complete response + partial response ) at 16 weeks will assessed based on Response evaluation criteria in solid tumors (RECIST) v1.1 criteria.

The overall Survival (OS) rate at 1 year is defined as the time from day 1 of study treatment until death as a result of any cause within one year of initiating study treatment.

-Progression Free Survival (PFS) rate at 6 months is defined as the time from day 1 of treatment until disease progression or death status measured 6 months after initiating study treatment. Progression events will be defined per Response evaluation criteria in solid tumors (RECIST) v1.1 criteria.

Procedures

Subjects in this trial will be given denosumab, 120 mg s.c. q4 weeks, starting on day 1 of study treatment. Additional loading doses of Denosumab will be administered on day 8 and day 22 ( after Amendment 1). Nivolumab, 480 mg will be administered intravenously (IV) every 4 weeks and initiated 21 days after the first dose of Denosumab is given. In subjects enrolled prior to Amendment 1 Pembrolizumab, 200 mg will be administered intravenously (IV) every 3 weeks and initiated 21 days after the first dose of denosumab is given. Combination therapy with both agents will continue as long as subjects benefit from therapy for up to 1 year. Study therapy will be discontinued for intolerable toxicity, disease progression or for other reasons at the discretion of the investigator. If subjects are not withdrawn prematurely then their last dose of study medications will be administered approximately 49 weeks after denosumab was initiated.

Arms:
arm 1: Subjects in this trial will be given denosumab every 4 weeks, starting on day 1 of study treatment. An additional loading dose of denosumab will be administered on day 8. Subjects who started Pembrolizumab (initiated 21 days after the first dose of denosumab is given) will continue to have it administered intravenously (IV) every 3 weeks. New subjects will receive Nivolumab administered intravenously (IV) every 4 weeks (initiated 21 days after the first dose of denosumab is given). Combination therapy will continue as long as subjects benefit from therapy for up to 1 year.

Interventions: intervention 1: Denosumab intervention 2: Pembrolizumab intervention 3: Nivolumab
Intervention descriptions: intervention 1: A dose of 120 mg will be administered as a subcutaneous (s.c.) injection every 4 weeks in the upper arm, upper thigh, or abdomen. Another loading dose of 120 mg s.c. denosumab will be administered on day 8. On days when denosumab is administered on the same day as pembrolizumab, the s.c. injection should be given after the infusion of pembrolizumab is completed. intervention 2: Pembrolizumab will be administered as standard of care following the institutional guidelines.The recommended dose of pembrolizumab is 200 mg administered as an IV infusion over approximately 30 minutes (range: 25 - 40 minutes) every 3 weeks until disease progression or unacceptable toxicity. intervention 3: Nivolumab will be given every four weeks at a dose of 480 mg to be administered as an IV infusion per institutional guidelines.

Protocol document:
Prot_SAP_000.pdf: 
LCCC1620 
Confidential 
Phase 2 Study of Denosumab and PD-1 Inhibitor in Subjects with Stage 
III/IV Melanoma 
Page i of 108 
 
 
 
SUMMARY OF CHANGES 
 
Protocol Amendment #4 
 
 
LCCC 1620: Phase 2 Study of Denosumab in Combination with a PD-1 Inhibitor in Subjects 
with Stage III/IV Melanoma 
AMENDMENT INCORPORATES: 
__  Editorial, administrative changes 
_X_  Scientific changes  
__  Therapy changes  
__   Eligibility Changes  
 
Rationale for amendment:  
The purpose for this amendment is to include the optional collection of tumor tissue from 
procedures that occurred prior to study enrollment as well as tumor tissue that may be collected 
after study completion. Often in clinical trials with rigorous translational endpoints where baseline 
tumor tissue is collected, the material is not sufficient or necrotic, impeding correlative studies. In 
these cases, any available archived material that is collected prior to study enrollment may be used 
in lieu of baseline tissue to assist in such studies. In addition, several patients as part of this study 
went on to undergo therapeutic lymph node dissections following study completion (or 
progression). Analyzing the tumor following end of study (or progression) is a great opportunity 
to assess change from baseline (effect of both denosumab plus nivolumab) and from day 21 (effect 
of denosumab alone). 
 
The reasons for requesting archived tumor blocks from procedures prior to study enrollment and 
following end of study (or progression) is to: 
A. Ensure that pretreatment tumor blocks are available in the event that the selected tumor 
block does not have sufficient and/ or and high quality tumor tissue available to perform 
tissue imaging and gene expression profiling studies. 
B. Analyze changes of the tumor tissue biomarker(s) under study treatment in earlier stages 
of melanoma (e.g. primary melanoma). 
C. Analyze changes of the tumor tissue biomarker(s) under study treatment from baseline 
(effect of both denosumab plus nivolumab) and from day 21 (effect of denosumab alone). 
 
The reasons for requesting archived tumor blocks that are from procedures that are conducted after 
completion of the study (or exit from the study due to tumor progression) are: 
A. For the archived tumor tissues that were generated as part of a surgery for tumor 
progression from the study (e.g. lymphadenectomy), we will assess the effect of treatment 
following combination with denosumab plus nivolumab.  




LCCC1620 
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III/IV Melanoma 
Page iii of 108 
 
 
 
Section 
7.3.2.1 
Addition of research tumor tissue collection for correlative study procedures.  
THE ATTACHED VERSION DATED July 7, 2022 INCORPORATES THE ABOVE 
REVISIONS  
 
 


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Confidential 
Phase 2 Study of Denosumab and PD-1 Inhibitor in Subjects with Stage 
III/IV Melanoma 
Page iv of 108 
 
 
 
 
PROTOCOL AMENDMENT 3 
 
 
 
LCCC 1620: Phase 2 Study of Denosumab in Combination with a PD-1 Inhibitor in Subjects 
with Stage III/IV Melanoma 
 
X  Editorial, administrative changes 
X    Scientific changes (IRB approval) 
     Therapy changes (IRB approval) 
 X Eligibility Changes (IRB approval) 
 
Rationale for amendment:  
This protocol amendment includes changes for consistency, clarifications, and eligibility to 
enhance patient enrollment. Clarifications were made to visit windows for weeks 17-49, and the 
use of glucocorticoids during the study was further clarified. Additional administrative updates 
include the removal of Case Western as a participating site, alignment of the timing of the first 
dose of nivolumab, and an editorial correction on enrollment numbers in the schema. 
 
Editorial/Administrative 
1. Case Western is removed as a participating site 
2. Section 1.2: Updated disease related information 
3. Section 1.8: Updated information related to denosumab and anti-PD1 agents.  
4. Section 4.1: The schema was updated to reflect the potential total enrollment number of n = 
28.  
5. Section 4.4.1 has been updated to clarify exception for glucocorticoid use during the study. 
6. In Section 6.1, the column in the Time and Events table labeled “Wks17-49 
(Wks22/28/34/40/46±2wks)” provides a visit window that is inconsistent with Footnote #1 (± 
7 days). The correct window is ± 7 days, as stated in Footnote 1, for those study visits. 
Therefore, the window is changed in Wks17-49 column in the Time and Events table in 
Section 6.1 from ±2wks to ±7 days. 
7. Section 6.1, footnote 1, the footnote contains a 7 week visit that is applicable to the 
pembrolizumab Time and Events table (Appendix B) but not applicable to nivolumab 
administration. The third sentence in Footnote 1 has been updated to reflect nivolumab 
administration. 
8. Section 6.1 Time and Events table; Footnote 12 is clarified to reflect the correct time to give 
the first dose of nivolumab; C2D2. 
 
Scientific Changes 
1. Section 6.1 Time and Events Table Footnote 17 and Section 6.3.2.1. In keeping with the 
change to the Criterion 3.1.16, information related to qualitative aspects of tissues have been 
removed. 


LCCC1620 
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Phase 2 Study of Denosumab and PD-1 Inhibitor in Subjects with Stage 
III/IV Melanoma 
Page v of 108 
 
 
 
Eligibility changes: 
1. Criterion 3.1.1.6: Removed qualitative measures surrounding archival tissue 
2. Criterion 3.2.1.2: The criterion is updated to clarify any dose of corticosteroids is 
unacceptable and subjects with brain metastases should be asymptomatic to participate in the 
study. Additionally, conditions were added as exceptions related to previously treated brain 
metastases that would allow for study entry. 
3. Criterion 3.2.1.11: Updated to exclude subjects with treatment of active immune disease in 
the past year rather than 2 years. 
 
THE ATTACHED VERSION DATED October 8, 2021INCORPORATES THE ABOVE REVISIONS 
ATTACH TO THE FRONT OF EVERY COPY OF PROTOCOL 
 
 
 


LCCC1620 
Confidential 
Phase 2 Study of Denosumab and PD-1 Inhibitor in Subjects with Stage 
III/IV Melanoma 
Page vi of 108 
 
 
 
 
PROTOCOL AMENDMENT 2 
 
 
 
 
LCCC 1620: Phase 2 Study of Denosumab in Combination with a PD-1 Inhibitor in Subjects 
with Stage III/IV Melanoma 
 
AMENDMENT INCORPORATES: 
X  Editorial, administrative changes 
    Scientific changes (IRB approval) 
X  Therapy changes (IRB approval) 
    Eligibility Changes (IRB approval) 
 
Rationale for amendment:  
This protocol amendment includes changes for consistency, and clarifications regarding timing 
of certain testing. 
 
Editorial/Administrative 
1. The timing of CBC with differential is listed correctly in Footnote 5 of the Time and Events 
Table in Section 6.1, but a discrepancy in the Table itself has been corrected by eliminating 
testing timepoints shown at C3D1 and C5D1. 
2. In Protocol Amendment 1, the time window of screening laboratory assessments was 
increased from 14 to 21 days (see “Summary of Changes”).  The timing of screening labs 
is listed correctly in Footnote 4 of the Time and Events Table in Section 6.1.  This change 
has been made consistent throughout the protocol.  The timing has now been corrected to 
21 days in the Inclusion Criteria (Section 3.1.1.9), as well as in Footnote 1 of the Time and 
Events Table in Section 6.1. 
3. In Section 3.2.1.5, an exclusion has been amended for consistency with exceptions in the 
inclusion criteria in Section 3.1.1.16. 
4. In the Time and Events Table in Section 6.1, the Archival Tissue/Tumor biopsies row 
included a reference to Footnote 17 at C2D1.  The reference has been corrected to Footnote 
18, which correctly clarifies that these samples are mandatory (as per Section 1.10.2). 
5. In the Time and Events Table in Section 6.1, the Archival Tissue/Tumor biopsies row 
included a reference to Footnote 18 at C5D1.  The reference has been corrected to Footnote 
19, which correctly clarifies that these samples are optional (as per Section 1.10.2). 
6. Protocol Amendment 1 simplified treatment by replacing pembrolizumab with nivolumab 
(see “Rationale”).  This change should be reflected throughout the protocol (for subjects 
enrolled after Amendment 1).  While the Time and Events Table in Section 6.1 correctly 
refers to nivolumab, Footnote 20 of the Time and Events Table referred to pembrolizumab.  
The references in the footnote and the relevant timepoints in the footnote have been 
updated.   


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III/IV Melanoma 
Page vii of 108 
 
 
 
7. Language in Section 9.6 regarding the process for amendments to the protocol has been 
updated to reflect the current process. 
8. Minor clarifying edits and format changes have been made in the document. 
 
 
Therapy changes: 
1. References to the time period for use of contraception have been corrected to 150 days in 
multiple sections including 3.1.1 and 4.5.2, in keeping with the longer time period indicated 
in the package inserts for the two relevant medications. 
2. In Section 4.2, the C12D1 Week 44 timepoint for PBMC blood draw is not needed and has 
been deleted. 
3. References to weight based dosage are not relevant and have been deleted from Sections 
4.2, 6.1, and 11.2.   
4. Reference to an anti-PD-1 agent has been clarified in Section 4.2. 
5. The Cycle 2 column in the Time and Events Table in Section 6.1 has been divided into two 
columns (D1 and D2) to clarify the order of events.  Verbiage has also been added to the 
footnotes for clarification.  Note:  This is the change from v1 to v1.1 of this Amendment 2. 
6. A heading in the Time and Events Table in Section 6.1 has been corrected from “C5 D8 – 
C13D8” to “C6 D1 – C13D1.” 
7. Regarding the Serum Chemistries row of the Time and Events Table (Section 6.1), footnote 
6 has been expanded to indicate that magnesium, calcium and phosphorus testing are 
required at C1D8. 
8. A footnote in the Endocrine row of the Time and Events Table in Section 6.1 has been 
eliminated to show that this testing is needed at each timepoint. 
9. The Urinalysis row in the Time and Events Table in Section 6.1 has been modified to show 
this testing at every other timepoint. 
10. A reference to footnote 19 in the denosumab row of the Time and Events Table in Section 
6.1 has been eliminated to indicate that certain timepoints are mandatory. 
11. A reference to footnote 19 in the PBMC row of the Time and Events Table in Section 6.1 
has been deleted to indicate that certain timepoints are mandatory. 
 
 
 
 
 
 
 
THE ATTACHED VERSION DATED July 26, 2019 INCORPORATES THE ABOVE REVISIONS 
ATTACH TO THE FRONT OF EVERY COPY OF PROTOCOL 
 
 


LCCC1620 
Confidential 
Phase 2 Study of Denosumab and PD-1 Inhibitor in Subjects with Stage 
III/IV Melanoma 
Page viii of 108 
 
 
 
 
PROTOCOL AMENDMENT 1 
 
 
 
 
LCCC 1620: Phase 2 Study of Denosumab in Combination with a PD-1 Inhibitor in Subjects 
with Stage III/IV Melanoma 
 
AMENDMENT INCORPORATES: 
X  Editorial, administrative changes 
X  Scientific changes (IRB approval) 
X  Therapy changes (IRB approval) 
X  Eligibility Changes (IRB approval) 
 
Rationale for amendment: A) Simplify treatment schedule by replacing anti-PD-1 agent 
pembrolizumab, which is administered every 3 weeks, with nivolumab which was recently 
approved by the FDA to be administered every 4 weeks, given that denosumab is administered 
every 4 weeks. Subjects who were enrolled into the study and started their treatment prior to 
approval of Amendment 1 will continue receiving pembrolizumab administered every 3 weeks. B) 
Move the Day 29 denosumab injection seven days earlier to allow for three loading doses of 
denosumab prior to initiating of anti-PD-1 therapy. C) Broaden eligibility criteria to allow for: (i) 
subjects with earlier than stage IV disease (e.g. bulky stage IIIB or IIIC disease; neoadjuvant 
administration), (ii) subjects who have progressed to stage IV melanoma following 1 year of 
adjuvant PD-1 inhibitors, (iii) subjects who have previously responded to PD-1 inhibitors for their 
stage IV disease by RECIST criteria and have discontinued PD-1 inhibitor due to clinical/subject 
preference or due to grade 1 or 2 toxicities, but now have progressed D) Update several portions 
of the protocol to accommodate transition to a multicenter study. 
 
Editorial/Administrative 
1. Updated Section 6.1, Section 6.2, Section 6.3 and Section 6.4 by adding bicarbonate (CO2) to 
the list of laboratory parameters assessed as part of comprehensive metabolic panel. 
2. Updated Section 7.3.3, Section 9.3, Section 9.4, Section 9.5.1, Section 9.5.2 and Section 9.6 to 
accommodate the transition to multicenter study. 
3. Updated Section 5.2 to revise pembrolizumab drug information. 
4. Added Section 5.3 with nivolumab drug information. 
5. Updated Section 4.2.1, Drug Dosing Schema, to accommodate use of nivolumab as an 
alternative anti-PD-1 agent following approval of Amendment 1.  
6. Added Section 4.3.2, Nivolumab Dose Modifications/Delays. 
 
 
Scientific changes: 


LCCC1620 
Confidential 
Phase 2 Study of Denosumab and PD-1 Inhibitor in Subjects with Stage 
III/IV Melanoma 
Page ix of 108 
 
 
 
1. Updated Section 6.1 to indicate that mandatory biopsy should performed 24 – 48 hours 
after third denosumab injection. 
2. Updated Section 1.8 to accommodate use of nivolumab as an anti-PD-1 agent following 
approval of Amendment 1. 
 
Therapy changes: 
1. Updated Section 1.1, Section 1.8, Section 1.9.2, Section 2.1.1, Section 4.1, Section 4.2 and 
Section 5.1.6 to reflect the addition of the third loading dose of denosumab on Day 22, 
prior to initiation of anti-PD-1 therapy. 
2. Updated Section 4.3.4, Pembrolizumab Dose Guidelines. 
3. Updated Section 6.6.2.1 to indicate that intrathoracic lesions are allowed to be biopsied, if 
deemed safe by the Principal Investigator. 
 
Eligibility changes: 
1. Revised inclusion criterion 3.1.1.4 to broaden eligibility for the study and indicate that 
subjects with histologically confirmed melanoma of mucosal primary may be enrolled into 
the study. 
2. Revised inclusion criterion 3.1.1.5 to broaden eligibility for the study and allow for 
subjects: (i) with earlier than stage IV disease (e.g. bulky stage IIIB or IIIC disease; 
neoadjuvant administration), (ii) who have progressed to stage IV melanoma following 1 
year of adjuvant PD-1 inhibitors, (iii) who have previously responded to PD-1 inhibitors 
for their stage IV disease by RECIST criteria and have discontinued PD-1 inhibitor due to 
clinical/subject preference or due to grade 1 or 2 toxicities, but now have progressed) at 
the discretion of the Principal Investigator. 
3. Revised inclusion criterion 3.1.1.9 to increase time window of screening laboratory 
assessments to 21 days. 
4. Revised inclusion criterion 3.1.1.9 to clarify that all but subjects with Gilbert’s syndrome 
must have a serum total bilirubin of ≤ 1.5 × ULN; subjects with Gilbert’s syndrome must 
have a serum total bilirubin of ≤ 2 × ULN to be eligible to participate in the clinical trial. 
5. Revised inclusion criterion 3.1.1.14 to broaden eligibility for the study and indicate that 
subjects who have grade ≤2 toxicities after prior radiation therapy that is stable for ≥3 
months) may be enrolled into the study. 
 
 
 
 
 
 
THE ATTACHED VERSION DATED DECEMBER 17, 2018 INCORPORATES THE ABOVE REVISIONS 
ATTACH TO THE FRONT OF EVERY COPY OF PROTOCOL 


LCCC1620 
Confidential 
Phase 2 Study of Denosumab and PD-1 Inhibitor in Subjects with Stage 
III/IV Melanoma 
Page x of 108 
LCCC 1620: Phase 2 Study of Denosumab in Combination with a PD-1 Inhibitor in 
Subjects with Stage III/IV Melanoma 
Short Title: Phase 2 Study of Denosumab plus a PD-1 Inhibitor in Subjects with Stage III/IV 
Melanoma 
Principal Investigator 
Stergios J. Moschos, MD 
Department of Medicine, Division of Hematology/Oncology 
University of North Carolina at Chapel Hill 
3116 Physicians Office Building 
170 Manning Drive, CB# 7305 
Chapel Hill, NC 27599-7305 
(919) 843-7713 (phone) | (919) 966-6735 (fax)
Email: moschos@med.unc.edu
Biostatistician  
Anastasia Ivanova, PhD 
3103C McGavran-Greenberg Hall 7420 
Email: aivanova@bios.unc.edu 
Phone: (919) 843-8086 
Clinical Protocol Office 
Lineberger Comprehensive Cancer Center 
The University of North Carolina at Chapel Hill 
450 West Drive, 3rd Floor, CB# 7295 
Chapel Hill, NC 27599-7295 
Multicenter Project Manager: 
CPOMultiCenter@med.unc.edu 
Sponsor:  Lineberger Comprehensive Cancer Center 
Funding Source: Amgen Inc. 
Protocol Version Date:  July 7, 2022 
Protocol Amendment:  4 
Version: 1.1 


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Phase 2 Study of Denosumab and PD-1 Inhibitor in Subjects with Stage 
III/IV Melanoma 
Page xi of 108 
 
 
 
LCCC 1620: Phase 2 Study of Denosumab in Combination with a PD-1 Inhibitor in 
Subjects with Stage III/IV Melanoma 
 
Short Title: Phase 2 Study of Denosumab and a PD-1 Inhibitor in Subjects with Stage III/IV 
Melanoma 
 
Principal Investigator 
Stergios Moschos, MD 
Department of Medicine, Division of Hematology/Oncology 
University of North Carolina at Chapel Hill 
3116 Physicians Office Building 
170 Manning Drive, CB# 7305 
Chapel Hill, NC 27599-7305 
(919) 843-7713 (phone) | (919) 966-6735 (fax) 
Email: moschos@med.unc.edu 
 
 
 
 
Signature Page 
 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable U.S. federal regulations and ICH guidelines. 
 
Principal Investigator (PI) Name:_______________________ 
 
PI Signature: ___________________ 
 
Date:____________________ 
 
Protocol Version Date:  July 7, 2022 
Protocol Amendment:  4 
Version: 1.1 
 
 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
i 
TABLE OF CONTENTS 
SUMMARY OF CHANGES ............................................................................................. i 
LIST OF ABBREVIATIONS ...........................................................................................v 
1.0 
BACKGROUND AND RATIONALE ..................................................................8 
1.1 
Study Synopsis ......................................................................................................8 
1.2 
Incidence and Current Standard of Care for Metastatic Melanoma ......................9 
1.3 
Immune Tolerance...............................................................................................11 
1.4 
Preclinical Evidence on the Role of the RANKL-RANK-AIRE Axis in 
Melanoma .......................................................................................................................16 
1.5 
Is the RANKL-RANK-AIRE Axis Clinically Relevant in Melanoma? .............17 
1.6 
Peripheral Effects of the RANK-RANKL System ..............................................19 
1.7 
Function of Thymus in Relation to Aging and Cancer; Facts and Unanswered 
Questions ........................................................................................................................20 
1.8 
Investigational Treatment – Denosumab Alone and with an Anti-PD-1 Agent .21 
1.9 
Rationale for the LCCC1620 Study Design ........................................................23 
1.10 
Correlative Studies ..............................................................................................24 
2.0 
STUDY OBJECTIVES .........................................................................................29 
2.1 
Co-primary Objectives ........................................................................................29 
2.2 
Secondary Objectives ..........................................................................................29 
2.3 
Exploratory Objectives ........................................................................................29 
3.0 
STUDY ENPOINTS..............................................................................................30 
3.1 
Co-primary Endpoints .........................................................................................30 
3.2 
Secondary Endpoints ...........................................................................................31 
3.3 
Exploratory Endpoints.........................................................................................31 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
ii 
4.0 
SUBJECT ELIGIBILITY ....................................................................................32 
4.1 
Inclusion Criteria .................................................................................................32 
4.2 
Exclusion Criteria ................................................................................................34 
5.0 
TREATMENT PLAN ...........................................................................................38 
5.1 
Schema ................................................................................................................38 
5.2 
Treatment Dosage and Administration ...............................................................39 
5.3 
Toxicities and Dosing Delays/Dose Modifications .............................................41 
5.4 
Concomitant Medications/Treatments ................................................................47 
5.5 
Rescue Medications and Supportive Care ...........................................................48 
5.6 
Other Modalities or Procedures...........................................................................51 
5.7 
Duration of Therapy ............................................................................................53 
5.8 
Duration of Follow Up ........................................................................................53 
5.9 
Removal of Subjects from Protocol Therapy ......................................................54 
5.10 
Study Withdrawal ................................................................................................54 
6.0 
DRUG INFORMATION ......................................................................................55 
6.1 
Denosumab ..........................................................................................................55 
6.2 
Pembrolizumab....................................................................................................57 
6.3 
Return and Retention of Study Medications .......................................................60 
6.4 
Nivolumab ...........................................................................................................60 
7.0 
EVALUATIONS AND ASSESSMENTS ............................................................63 
7.1 
Time and Events Table ........................................................................................63 
7.2 
Follow-up Assessments .......................................................................................65 
7.3 
Correlative Studies Procedures ...........................................................................66 
7.4 
Assessment of Safety ..........................................................................................68 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
iii 
7.5 
Assessment of Efficacy .......................................................................................69 
8.0 
ADVERSE EVENTS ............................................................................................73 
8.1 
Definitions ...........................................................................................................73 
8.2 
Documentation of non-serious AEs or SARs ......................................................75 
8.3 
SAEs or Serious SARs ........................................................................................75 
8.4 
Data and Safety Monitoring Plan ........................................................................78 
9.0 
STATISTICAL CONSIDERATIONS ................................................................79 
9.1 
Study Design/Study Endpoints ...........................................................................79 
9.2 
Sample Size and Accrual.....................................................................................82 
9.3 
Data Analysis Plans .............................................................................................82 
10.0 STUDY MANAGEMENT ....................................................................................84 
10.1 
Institutional Review Board (IRB) Approval and Consent ..................................84 
10.2 
Required Documentation.....................................................................................84 
10.3 
Registration Procedures.......................................................................................84 
10.4 
Data Management and Monitoring/Auditing ......................................................85 
10.5 
Adherence to the Protocol ...................................................................................85 
10.6 
Amendments to the Protocol ...............................................................................87 
10.7 
Record Retention .................................................................................................87 
10.8 
Obligations of Investigators ................................................................................88 
11.0 REFERENCES ......................................................................................................89 
12.0 APPENDICES .......................................................................................................94 
12.1 
Appendix A. ECOG Performance Status ............................................................94 
12.2 
Appendix B. Time and Events Table (For Subjects Receiving Pembrolizumab)
 
95 
 


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LIST OF ABBREVIATIONS  
AE 
Adverse event 
AIRE 
Autoimmune regulator 
AJCC 
American Joint Committee in Cancer 
ALC 
Absolute lymphocyte count 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
AST 
Aspartate aminotransferase 
β-HCG 
Beta–human chorionic gonadotropin 
BRCA1 
Breast cancer gene 1 
CBC 
Complete blood count 
CL 
Chloride 
CLIA 
Clinical Laboratory Improvement Amendments 
CO2 
Bicarbonate 
CPO 
Clinical protocol office 
CR 
Complete response 
CRA 
Clinical Research Associate 
CrCl 
Creatinine clearance 
CRF 
Case report form 
CT 
Computer tomography 
CTLA 
Cytotoxic T lymphocyte-associated 
CYP 
Cytochrome P450 
D1 
Day 1 
DC 
Dendritic cell 
dL 
Deciliter 
DSMC 
Data safety monitoring committee 
ECI 
Event of clinical interest 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic case report form 
ELISA 
Enzyme-linked immunosorbent assay 
FACS 
Fluorescence-activated cell sorting 
FDA 
Food and Drug Administration 
FFPE 
Formalin-fixed paraffin-embedded 
GCP 
Good clinical practice 
GFR 
Glomerular filtration rate 
H&E 
Hematoxylin & Eosin 
Hgb 
Hemoglobin 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human immunodeficiency virus 
Hr 
Hour 
IB 
Investigator’s Brochure  
ICP 
Immune checkpoint proteins 
ID 
Identification 
IDS 
Investigational drug service  
IF 
Immunofluorescence 
IHC 
Immunohistochemistry 
IMGF 
Immune Genomics Facility 
INR 
International normalized ratio 
IRB 
Institutional Review Board 
IU 
International unit 
IV 
Intravenous 
K 
Potassium 
Kg 
Kilogram 


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LDH 
Lactate dehydrogenase 
MDSC 
Myeloid-derived suppressor cell 
Mg 
Milligram 
MHC 
Major histocompatibility class 
Min 
Minute 
mL 
Milliter 
MRI 
Magnetic resonance imaging 
Na 
Sodium 
NCI-CTCAE 
National Cancer Institute – Common Terminology Criteria for Adverse 
Events 
NE 
Nonevaluable 
NK 
Natural killer 
ONJ 
Osteonecrosis of the jaw 
OPG 
Osteoprotegerin 
ORR 
Overall response rate 
OS 
Overall survival 
OTC 
Over the counter 
PBMC 
Peripheral blood mononuclear cells 
PCR 
Polymerase chain reaction 
PD 
Progressive disease 
PD-1 or PD-L1 
Programmed death-1 or Programmed death ligand 1 
PE 
Physical Exam 
PET 
Positron Emission Tomography 
PFS 
Progression-free survival 
PFT 
Pulmonary function test 
Pgp 
P-glycoprotein 
PR 
Partial response 
PT 
Prothrombin time 
PTT 
Partial thromboplastin time 
Qd 
Quaque die (once daily) 
Q3 weeks 
Every 3 weeks 
RANK 
Receptor activator for NFκB 
RANKL 
Ligand for Receptor activator for NFκB 
RECIST 
Response evaluation criteria in solid tumors 
RIP3 
Receptor interacting protein 
RNA 
Ribonucleic acid 
RTE 
Recent thymic integrants 
SAE 
Serious adverse event 
s.c. 
subcutaneous 
SD 
Stable disease 
sjTREC 
signal joint T-cell receptor excision circles 
SmPc 
Summary of product characteristics 
SNPs 
Single nucleotide polymorphism 
SUSAR 
Serious unexpected adverse reaction 
T4 
Thyroxine 4 
TAM 
Tumor-associated macrophages 
TCR 
T-cell receptor 
T1DM 
Type 1 diabetes mellitus 
TEC 
Thymic epithelial cells 
TIL 
Tumor infiltrating lymphocyte 
TNFSFR 
Tumor necrosis factor receptor superfamily 
TPL 
Translational pathology lab 
Treg 
T regulatory  
TRP-1 
Tyrosinase related protein-1 
TSH 
Thyroid stimulating hormone 
ULN 
Upper limit of normal 


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UNC 
University of North Carolina 
Wk 
Week 
WOCBP 
Woman of childbearing potential 
 
 
 


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1.0 
BACKGROUND AND RATIONALE 
1.1 
Study Synopsis 
This is a multicenter open-label, single-arm, phase 2 study designed to investigate 
the pharmacodynamic and antitumor effects of denosumab alone and in 
combination with an anti-PD-1 agent (pembrolizumab in subjects enrolled prior to 
Amendment 1 and nivolumab in subjects enrolled after Amendment 1) in subjects 
with unresectable PD-1/PD-L1 inhibitor-naïve regional and distant metastatic 
melanoma (AJCC stage III/IV). The pharmacodynamic and antitumor effects will 
be investigated by performing translational research on peripheral blood and tumor 
tissue collected before and during denosumab alone and in combination with anti-
PD-1 treatment (details below). Up to 25 subjects will receive denosumab at the 
FDA approved dose (120 mg s.c. every 4 weeks with additional loading doses of 
denosumab 120 mg s.c. on day 8 and day 22 following Amendment 1) concurrently 
with either nivolumab at the recently FDA-approved every 4 week dose (480 mg 
IV q4wks in subjects enrolled following Amendment 1), or pembrolizumab at the 
FDA-approved dose (200 mg IV every 4 weeks in subjects enrolled prior to 
Amendment 1) for up to a year based on clinical benefit. Subjects who continue to 
have clinical benefit after a year of treatment may continue therapy with PD-1/PD-
L1 inhibitor as per standard of care (See Study Schema). 
 
The study has co-primary objectives; one being to assess the pharmacodynamic 
effects (immunomodulatory and/or antitumor) of denosumab alone and the other 
being to assess the pharmacodynamic effects following combination therapy with 
denosumab and an anti-PD-1 agent. To investigate any potential direct antitumor 
and indirect immunomodulatory effects of denosumab alone, concomitant anti-PD-
1 treatments will not start until day 22 of study treatment initiation, after three doses 
of denosumab have been given on day 1, day 8, and day 22. In fact, mandatory 
tumor biopsies and peripheral blood will be collected on day 22, immediately prior 
to the first anti-PD-1 agent infusion. Blood and tumor samples collected on day 22 
will allow us to determine the denosumab-alone effect compared to peripheral 
blood samples and archived or fresh tumor tissue collected at baseline. Multi-
parameter flow cytometry of peripheral blood mononuclear cells (PBMCs), ELISA 
assays of serum, and immunohistochemistry (IHC) and immunofluorescence (IF) 
analyses of tumor tissue are the methods that will be utilized for correlative studies.  
 
The secondary co-primary endpoint of the study includes assessment of the 
pharmacodynamic effects of denosumab and anti-PD-1 agent combination therapy 
in peripheral blood samples collected during the study (i.e., at weeks 16, 28, and 
40) for comparison with denosumab alone. Moreover, an optional tumor biopsy 
may be obtained at week 16. Secondary endpoints include assessments to determine 
the safety of the combination in subjects with metastatic melanoma, plus 
measurements of the clinical benefit of denosumab in combination with an anti-
PD-1 agent based on response rate, per Response Evaluation Criteria in Solid 
Tumors version 1.1 (RECIST v1.1) criteria at week 16, progression-free survival 


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(PFS) rate at 6 months of combination therapy and overall survival (OS) rate at 1-
year of study treatment.  The safety of the combination will also be evaluated per 
NCI Common Terminology Criteria for Adverse Events (NCI-CTCAE). Pre-
defined Pocock-stopping criteria will dictate early trial termination, if excessive 
toxicity or other adverse effects (AE) of clinical interest are observed with this 
combination.              
1.2 
Incidence and Current Standard of Care for Metastatic Melanoma  
The American Cancer Society estimates that approximately 106,110 new cases of 
melanoma and 7,180 melanoma-related deaths will occur in the United States in 
20211. Overall, the lifetime risk for melanoma is about 2.5%. While the average 
age of patients diagnosed with melanoma is 63 years, around the time that immune 
system may be compromised due to aging, a concerning statistic is that melanoma 
is also a common cancer in young adults. Early detection and treatment 
significantly impacts long-term prognosis as OS drops precipitously for patients 
diagnosed with melanomas that are locally advanced and/or distant metastatic. The 
5-year OS rate is only about 15% to 20% for patients diagnosed with stage IV 
melanoma in comparison to patients with early stage tumors (Stage IA to IIB) with 
5-year survival rates in the 87% to 99% range2. Unfortunately, metastatic cutaneous 
melanoma remains difficult to treat despite the introduction of novel therapies for 
this disease in recent years. 
 
Significant advances have occurred in the treatment of metastatic cutaneous 
melanoma with ten new treatments having been approved by the US FDA over the 
last 10 years. These novel treatments include small molecule inhibitors that target 
BRAF and MEK signaling pathways and monoclonal antibodies directed against 
the immune checkpoint proteins (ICP) PD-1 and CTLA-4 3,4. ENREF 3Targeted 
therapies with BRAF inhibitors are well studied in patients with BRAFV600E-
mutant melanoma, For example, the 3-year OS rate of patients with BRAFV600-
mutant melanoma who received dabrafenib or vemurafenib  (both are BRAF 
inhibitors) alone was 41% and 21-31%, respectively whereas the median OS of 
concurrent inhibition of BRAF and MEK, using dabrafenib and trametinib or 
encorafenib (another BRAF inhibitor) plus binimetinib (a MEK inhibitor) was 25.9 
months and  33.6 months, respectively 5-7. However, only ~50% of melanoma 
patients harbor this mutation. More recently, treatment of patients with NRASQ61-
mutant melanoma with binimetinib, another MEK inhibitor, significantly 
prolonged PFS compared to standard chemotherapy; however the clinical benefit 
was relatively small8._ENREF_7 
 
Immunotherapies targeting co-inhibitory ICP CTLA-4 (ipilimumab) and PD-1 
(nivolumab, pembrolizumab) have demonstrated considerable clinical benefit 
measured by durable PFS and OS. Clinical benefit from single-agent PD-1 
inhibitors has been significantly greater compared to that conferred by CTLA-4 
inhibitors (ipilimumab), based on randomized phase III studies 9,10. Furthermore, 
PD-1 inhibitors are better tolerated than ipilimumab alone and with a lower 
incidence of serious adverse events (SAEs). Interestingly, concurrent blockade of 


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PD-1 and CTLA-4 has been associated with overall response rates (RR) up to 58%, 
as measured by RECIST v1.1 criteria; however, this is counterbalanced by 
increased risks of up to 55% in treatment-related SAEs 9 and lack of overall survival 
clinical benefit compared to nivolumab alone_ENREF_11.11 A clinical trial using 
a lower dose of ipilimumab administered on a less frequent dosing schedule, 
suggests that the major driver of SAEs seen with concurrent PD-1/CTLA-4 
blockade regimens is due to ipilimumab_ENREF_12.12  Overall, the high frequency 
of serious and life-threatening AEs observed with concurrent PD-1 and CTLA-4 
blockade has limited the use of this regimen in community oncology settings 
despite its indisputable superior clinical benefit. Nevertheless, the superiority of 
concurrent dual CTLA-4 and PD-1 blockade over single target blockade suggests 
that multiple etiologies may simultaneously impair host immune response. 
Identification of novel immune-based targets that are dysregulated in cancer and 
are amenable to drug manipulation may potentially identify combination 
immunotherapeutic strategies that match the clinical benefit conferred by 
concurrent CTLA-4/PD-1 blockade without the intolerable side effects. 
1.2.1 Future Directions for Treating Metastatic Melanoma 
Further advances in the treatment of metastatic melanoma are focusing upon: (a) 
understanding the optimal sequence of targeted therapies versus immunotherapies 
in patients with BRAFV600 and NRASQ61 mutations (NCT02224781, EA6134), 
(b) identifying patient subgroups that benefit from combination CTLA-4/PD-1 
blockade as opposed to single-agent PD-1 blockade, (c) administering targeted 
therapies with PD-1/PD-L1 pathway inhibitors simultaneously, in particular for 
patients with BRAFV600-mutant melanoma (e.g. NCT02130466, KEYNOTE-
022), (d) combining PD-1/PD-L1 pathway inhibitors with immunotherapies other 
than CTLA-4 or cytokines (e.g. IL-2, interferons) based on better understanding of 
novel mechanisms of immune dysfunction, (e) testing novel targeted therapies for 
patients with non BRAFV600-mutant melanoma. Approach (d) is crucial given the 
high degree of toxicity of combined nivolumab and ipilimumab and the lack of 
predictive biomarkers of response, despite the indisputable durable clinical benefit. 
Therefore, PD-1/PD-L1-based treatments with comparable efficacy to nivolumab-
ipilimumab, and a better toxicity profile may hold greater promise than concurrent 
nivolumab-ipilimumab.  
 
Our proposed study is in line with priority (d) listed above. We have identified that 
RANKL-mediated central immune tolerance is clinically relevant in melanoma, 
based on both preclinical observations and clinical findings (see Sections 1.4 and 
1.5)13. Thus, we propose to conduct this exploratory phase 2 study of anti-PD-1 
agent (nivolumab administered at the FDA-approved dose 480 mg every 4 weeks 
in subjects enrolled after Amendment 1, or pembrolizumab administered at the 
FDA-approved dose 200 mg every 3 weeks in subjects enrolled prior to 
Amendment 1) in combination with denosumab administered at the FDA-approved 
dose (120 mg s.c. every 4 weeks with the addition of a loading dose of 120 mg 
administered on day 8 and day 21) in subjects with unresectable regional or distant 
metastatic melanoma (AJCC stage III/IV). We predict that this treatment 


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combination will be safe, well tolerated and may lead to antitumor responses that 
are significantly higher than those observed with a single anti-PD-1 agent. 
 
To better understand the translational approach taken in our proposed study of 
denosumab-anti-PD-1 agent combination therapy in subjects with metastatic 
melanoma, it is important to know how immune function and dysregulation relate 
to immunotherapeutic treatment strategies for this disease. The following sections 
provide background on the salient topics of immune tolerance, normal T cell 
development, thymus function, and the role of RANK-RANKL-AIRE in thymus 
function with a particular emphasis on how these phenomena may influence the 
development and treatment of melanoma. 
1.3 
Immune Tolerance 
Immune tolerance is a state of immune system unresponsiveness to antigens that 
may otherwise enable immune response 14. This state of tolerance is contrasted with 
the conventional immune-mediated elimination of ‘non-self’/foreign antigens 
important for normal physiology (e.g. pregnancy). The balance between tolerance 
and immune-mediated elimination of ‘non-self’/foreign antigens comes with some 
crucial negative tradeoffs. For example, deficits in immune tolerance can lead to 
autoimmune diseases, infections, and cancer. Immune tolerance is classified as 
central or peripheral, depending on the anatomic location where tolerance is 
originally induced (e.g. thymus/bone marrow versus peripheral tissues/lymph 
nodes). The sections below elaborate on the role of immune tolerance in cancer, 
with particular emphasis on the most well studied peripheral type, and how recent 
advances in understanding of the mechanisms of central immune tolerance may be 
clinically relevant for cancer and even provide opportunities for novel anticancer 
therapies. 
 


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Figure 1. Central and peripheral immune tolerance in cancer. The complexity and the 
various immune cell subset involved, underlies multiple levels of regulation that can be 
potentially exploited for therapeutic intent. It also implies why single immunotherapy 
approaches may not be as effective as combination strategies, such as concurrent PD-1 and 
CTLA-4 blockade. Adapted from 4. 
 
1.3.1 Peripheral Immune Tolerance is the Most Well Studied and Clinically 
Applicable Immunoregulatory Mechanism 
Peripheral immune tolerance is key in preventing over-reactivity of the immune 
system to various environmental antigens (allergens, gut microbes, etc.). 
Mechanisms of peripheral immune tolerance in cancer are complicated and diverse, 
but have been extensively studied.  Large-scale analysis of cutaneous melanoma 
tumor samples shows that in approximately 50% of melanomas, tumor-infiltrating 
lymphocytes (TIL) express both co-inhibitory and co-activating signals, a sign of 
an inability of immune cells to clear non-self-antigens. More specifically, as long 
as the ‘non-self’ antigen cannot be effectively eliminated, early upregulation of co-
activating ICP that regulate cell proliferation, survival, cytotoxicity, type I 
interferon production, and activation is progressively followed by compensatory 
upregulation of co-inhibitory ICP that render immune cells more prone to apoptosis 
and anergy. In advanced stages of peripheral immune tolerance, TILs become 
tolerant of the nearby tumor cells in a symbiotic state that favors the latter 15. As 
shown in Figure 1 peripheral immune tolerance involves expression of various co-
inhibitory ICP ligands and/or their cognate receptors by cancer cells themselves 
and/or other immunoregulatory populations, such as T regulatory cells (Treg), 
myeloid-derived suppressor cells (MDSC), and dendritic cells (DC). CTLA-4 and 
PD-1 are two of the most extensively studied co-inhibitory ICP targets in melanoma 


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and other cancers and their targeted inhibition is now an FDA-approved treatment 
strategy for various cancers. A number of clinical trials are underway that 
simultaneously target ICPs other than CTLA-4 and PD-14 _ENREF_5as a strategy 
for effectively inhibiting peripheral immune tolerance while keeping toxicity to a 
minimum. 
1.3.2 Central Immune Tolerance is Insufficiently Explored in Cancer 
Central tolerance is the main mechanism used by the immune system for 
discriminating ‘self’ from ‘non-self’. Central tolerance relies on the continuous 
production of early precursor T cells by the bone marrow, followed by their 
maturation within the thymus. Multiparameter flow cytometry applied to PBMC 
allows us to follow the distinct immune cell subsets that are prevalent during each 
maturation stage without need for invasive biopsies to the thymus, which are nearly 
impossible in the adult life due to thymus involution. Nevertheless, few studies 
have investigated the abundance and functional status of these and other relevant 
immune cell subsets during physiologic (e.g. aging) and pathologic conditions (e.g. 
cancer) and the potential role that changes in these immune cell subsets may play 
in the etiopathogenesis of various diseases, including cancer. To understand the role 
of central immune tolerance in cancer, it is important to review the sequential steps 
and the corresponding regulatory feedback loops involved in the generation of 
mature T cells (Figure 2; also 16).  
1.3.2.1 Normal T Cell Development 
Precursor T cells are derived from bone marrow stem cells.  They migrate from the 
bone marrow to the thymus cortex as CD3-CD4-CD8- (double negative) cells for 
further processing. In the steps following T cell maturation within the thymus, the 
physical interaction between precursor T cells with cortical and medullary thymic 
epithelial cells (TEC) is crucial; therefore, regulation of TEC development, 
differentiation, and self-tolerance by various endocrine signals as well as other 
cytokines may significantly affect the type and repertoire of both effector immune 
response as well as peripheral tolerance. These double negative cells first undergo 
T-cell receptor (TCR) re-arrangement, which is an important maturation step 
towards diversification of T cells to different clones that will later on recognize 
distinct peptide epitopes. The extent of TCR re-arrangement among T cells, the 
surrogate for T-cell clonality repertoire and therefore epitope diversity, can be 
assayed by measuring signal joint TCR excision circles (sjTREC), namely small 
circles of DNA generated by T cells during their passage through the thymus as 
they rearrange their TCR genes 17,18. In fact, sophisticated laboratory assays that 
precisely quantify TREC in the peripheral blood can be performed in clinical 
laboratory improvement amendments (CLIA) certified laboratories (e.g. Mayo 
Laboratories, Duke) and have been infrequently used as a diagnostic tool to monitor 
thymic output in certain physiologic states (e.g. aging) and diseases (e.g. T-cell 
reconstitution following bone marrow transplantation, autoimmune diseases). 


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Figure 2. Schematic presentation of thymic input, thymic output, and intrathymic 
T cell development. Emphasis on negative selection (red-filled cells) and the role of 
RANK-RANKL-AIRE axis. Abbreviations: TCR, T-cell receptor; mTEC, medullary 
thymic epithelial cells; RTE, recent thymic emigrants; Treg, T regulatory cells. 
 
Following TCR rearrangement, double negative precursor T cells become 
CD4+CD8+ (double positive) cells and undergo positive selection within the 
thymus cortex, only if they bear a TCR self-peptide complex that can interact 
with major histocompatibility class (MHC) class I or MHC class II receptors on 
the surface of TEC.  Cells that are unable to bind to MHC receptors undergo 
apoptosis.  At this point, T cells are either CD4+ or CD8+ (single negative), 
depending on which MHC class they are able to bind to during positive selection, 
and enter the thymic medulla for further processing.  Of the T cell clones that 
undergo positive selection, those bearing TCR:self-peptide complexes that bind 
too strongly with self MHC are clonally deleted whereas the remaining are 
released from the thymus as single positive (CD4-CD8+ or CD4+CD8-) recent 
thymic 
emigrants 
(RTE). 
RTE 
are 
essentially 
the 
naïve 
T-cells 
(CD45RA+CD45RO-) that travel to secondary lymphoid organs (i.e. spleen, 
lymph nodes) and become effector T cells capable of proliferation and activation 
following encounter with distinct antigens.  Phenotypically, they express the 
following 
membrane 
markers: 
CD4+ 
or 
CD8+ 
CD45RO-
CD45RA+CD62L+CD31+ 19 and their clonal diversity can be measured by 
TREC, as mentioned above. TREC are diluted as T cells divide, so high levels 
indicate that a cell is a RTE (reviewed in 20,21). 
1.3.2.2 
Regulation of Thymic Function; the Particular Role of RANKL and AIRE 
It is important to emphasize that the process of T-cell development and maturation 
is highly regulated by various endocrine and paracrine signals. Understanding these 


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positive and negative feedback loops and the precise anatomic locations within the 
thymus where they exert their effect (cortex versus medulla) is important, because 
various normal physiologic and pathologic states may affect thymic function via 
changes in the abundance of these signals. For example, keratinocyte growth factor 
and growth hormone enhance proliferation of both cortical and medullary TEC.  
Negative regulators include glucocorticoids that cause decreased proliferation in 
cortical TEC (cTEC) and increase apoptosis in medullary TEC (mTEC) with the 
immature double positive thymocytes being the most susceptible 22. Androgens and 
estrogens at high levels (e.g. pregnancy) suppress thymic output of naïve T cells 
via effects on both TEC subsets as well as bone marrow stromal cells. In contrast, 
physiologic levels of estrogens are fundamental for thymus development. 
Identification of these factors as regulators of thymic development is clinically 
relevant, because it may in part explain several long-standing clinical observations, 
such as: 
(a) why females usually have better prognosis than males across various 
cancer types after adjusting for age, stage, and duration of follow-up 23? 
(b) why advanced age may be an adverse prognostic factor across various 
cancers (e.g. small-cell lung cancer, melanoma, bladder, ovarian), after 
adjusting for sex, cancer stage, performance status, and various other 
comorbid 
factors24? 
(c) why females are more prone to autoimmune diseases than men 25? 
(d) why chemical castration in patients with prostate cancer increases 
circulating naïve T-cells 26? 
(e) why obese males have greater clinical benefit to both targeted therapies 
and immune checkpoint inhibitors compared to all other groups27? 
 In addition to hormonal factors, various members of the tumor necrosis factor 
(TNF) receptor superfamily (TNFSFR) play a significant role in regulating mTEC 
development and function. More specifically, mTEC express three principal 
TNFSFR members, the receptor activator for NFκB (RANK), CD40, and the 
lymphotoxin beta receptor (LTβR). Ligands for the three corresponding receptors 
(TNFSFRL), RANKL, CD40L, and lymphotoxin α/β are normally expressed by 
thymocytes and bind to their respective receptors on mTEC to initiate signaling and 
activation of classical and non-classical NFκB pathways 28. In the case of RANKL-
RANK, this pathway positively regulates expression of the autoimmune regulator 
(AIRE) in mTEC. AIRE is a transcription factor that regulates expression of a large 
set of downstream genes; 45% encode peripheral tissue antigens, which largely 
represent auto-antigens expressed by diverse tissues and organs. Therefore, 
exposure of differentiating thymocytes to a large variety of peripheral tissue 
antigens ensures negative selection of highly self-reactive nascent T-cell clones and 
prevents autoimmune diseases. As expected, loss-of-function mutations in the 
human Aire gene causes a recessive autoimmune syndrome, termed autoimmune 
polyendocrinopathy-candidiasis-ectodermal dystrophy 29.  


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1.4 
Preclinical Evidence on the Role of the RANKL-RANK-AIRE Axis in 
Melanoma 
Pioneering research by Dr. Maureen Su and colleagues from our institution has 
shown that molecular targeting of AIRE in mTEC can induce antitumor responses 
in melanoma syngeneic mouse models. More specifically, Dr. Su and colleagues 
have shown that the mechanism is immune-mediated and tumor antigen-specific; 
Aire-deficient mice have increased escape of specific T cells against melanoma-
associated antigens (e.g. tyrosinase-related protein-1, TRP-1) from negative 
selection.  This results in increased numbers of melanoma antigen-specific CD8+ 
cells infiltrating tumors, which in turn suppresses melanoma growth and improves 
survival 30. Dr. Su and colleagues went on to show that pharmacologic blockade 
using an antibody targeting RANKL in melanoma-bearing syngeneic mice confers 
an antitumor effect that is similar to what was seen with Aire-deficient melanoma-
bearing mice 31. Given the availability of RANKL inhibitors in the clinic, these 
results point towards the potential role of RANKL blockade as a novel 
immunotherapeutic strategy in melanoma. 
 
More recently, Dr. Su and colleagues from our institution have shown that 
simultaneous targeting of CTLA-4 and RANKL in syngeneic melanoma mouse 
models results in antitumor responses that are superior compared to either treatment 
alone (Figure 3, and 13). The mechanism of action is novel, because it does not 
involve significant changes in the activation status of tumor-infiltrating CD8+ T 
cells, but rather upregulation of tumor-infiltrating, proliferating (Ki67+), killer-like 
receptor subfamily G member 1 positive (KLRG1+) –a unique CD4+ T cell 
population that expresses high levels of cytotoxicity-associated genes 32– and 
cytolytic (granzyme B+) ‘effector’ CD4+ T cells. Further analysis of the immune-
mediated changes in the Aire-deficient or loss-of-function AireG228W mice shows 
that the expression of the ICP, CTLA-4, PD-1, and OX-40, is increased in CD4+ T 
effector cells, whereas CTLA-4 was not significantly changed in CD4+FoxP3+ 
Tregs (Figure 4 and 13). Furthermore, Dr. Su and colleagues have shown that Aire 
deficiency and CTLA-4 blockade have additive effects by increasing the frequency 
of T cells that are reactive against the self/melanoma antigen TRP-1 that includes a 
trend towards increased frequency of T cells reactive against other self/melanoma 
antigen, TRP-2 and gp100, but not towards the irrelevant foreign antigen, 
ovalbumin 13. In summary, Aire deficiency upregulates a unique population of 
melanoma-associated cytotoxic CD4+ T cells that are activated and express various 
co-inhibitory and co-stimulatory ICP, including CTLA-4 and PD-1. It is perhaps 
the upregulation of co-inhibitory ICP in those unique CD4+ cells, including CTLA-
4 that, if inhibited by CTLA-4 antibodies, induces antitumor responses that are 
superior to Aire deficiency alone or CTLA-4 blockade alone.  
 


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Figure 3. Anti-RANKL antibody enhances the immunotherapeutic effects of aCTLA-
4 antibody monotherapy (A) or combination aCTLA-4 and aPD-1 therapy (B) in 
melanoma-bearing mice. Left panel. Schema of combination treatment strategy to block 
central and peripheral T cell tolerance mechanisms. Anti-RANKL antibody or isotype 
control antibody was injected every other day for 11 days, followed by subcutaneous B16 
melanoma injection (1×105 cells) on Day 18. GVAX with or without anti-CTLA-4 
antibody (A) or combination anti-CTLA-4 plus anti-PD-l1 antibody was given on days 3, 
6, and 10 following melanoma cell injection. Host survival curves in GVAX treated mice 
receiving indicated combinations of antibody(ies) therapy. n=5 for each group. *p<0.05. 
Tumor-infiltrating lymphocytes (TIL) were harvested on Day 19 following B16 melanoma 
inoculation. 
 
1.5 
Is the RANKL-RANK-AIRE Axis Clinically Relevant in Melanoma? 
Several lines of evidence suggest that AIRE is clinically relevant in melanoma. 
Firstly, analysis of the cutaneous melanoma TCGA dataset 33 suggests increased 
expression of various melanoma-associated and cancer testis antigens across all 
melanoma subsets (Figure 5). Secondly, AIRE expression and peripheral tissue-
specific antigen expression are highly variable among individuals 34. This may be 
explained by the fact that several single nucleotide polymorphisms (SNPs) can be 
found in humans. Although the impact of most of these SNP on Aire RNA stability 
and protein function is unknown, a handful of SNPs have been associated with 
decreased AIRE mRNA stability, and presumably decreased AIRE protein 
expression 35. We postulate that differences in AIRE expression among humans 
may explain why certain individuals may be more prone to autoimmune diseases 
than others. Interestingly, distinct SNPs in the Aire gene were more frequent in 
healthy subjects as opposed to patients with melanoma diagnosis, presumably due 
to decreased stability of the AIRE mRNA 35. 
 


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To assess whether certain Aire polymorphisms, which presumably lead to 
decreased levels of Aire, and therefore increases in the frequency of autoreactive 
clones, predict response to ipilimumab, Dr. Su and colleagues genotyped 79 
patients with metastatic cutaneous melanoma who participated in the E1608 study36 
for 5 Aire polymorphisms (rs1800522, rs2075876, rs56393821, rs1800520, and 
rs1055311). All 79 patients received ipilimumab alone. The rs1055311 TT 
polymorphism, which is associated with protection against melanoma development 
35, was present in 6/79 patients (7.6%). Interestingly, patients with the TT SNP had 
an increased incidence (50%) of complete or partial responses (CR, PR) to 
ipilimumab13, which is approximately 3 times higher than the antitumor responses 
seen with ipilimumab in large phase III studies 9. These results suggest that 
although the incidence of patients with metastatic melanoma with Aire SNPs that 
may lead to a higher frequency of autoreactive clones is low, it is those patients 
who perhaps derive the greatest benefit from ipilimumab treatment. It is therefore 
tempting to hypothesize that targeting the pathway that regulates AIRE expression 
in mTEC, RANKL-RANK, may further increase the number of patients with 
metastatic melanoma who express autoreactive T cell clones, presumably against 
melanoma-associated and cancer testis antigens, which in combination with CTLA-
4 blockade may increase antitumor response. In fact, durable responses following 
treatment with ipilimumab plus denosumab have already been reported in patients 
with metastatic melanoma 37. 


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Figure 5. mRNA expression of various self-antigens (cancer testis, CT; and melanoma 
associated antigens) in cutaneous melanoma samples as part of the RNAseq analysis 
of the TCGA 329 tumor dataset33. Antigens included are of the MAGE family (A1, A4, 
A5, A9B, A10, A11, B1, B2, C1, C2, D4, D4B, L2,), PAGE family (1, 2, 2B, 5), cancer 
testis (CT) family (45A1, AG1B, AG2, AGE9), VCX family (VCX, 3A, 3B), FAM133A, 
XAGE1D, TPTE, CXORF48. Median expression was calculated after rank ordering (with 
1 being the highest expression) in relation to the median expression across all three 
subtypes. Courtesy of Dr. Katherine Hoadley, Ph.D. 
 
1.6 
Peripheral Effects of the RANK-RANKL System 
RANKL (also known as TRANCE/TNFSF11/ODF) along with its signaling 
receptor, RANK, and the soluble decoy receptor, osteoprotegerin (OPG), were 
discovered in the late 1990s as an important TNF superfamily system primarily 
involved in DC viability and function as well as in bone homeostasis, via regulation 
of osteoclasts. In its simplified form, RANKL is upregulated in T cells upon TCR 
activation (more in memory CD4 (Th1>Th2) than in memory CD8 cells) with 
kinetics much slower than the CD40-CD40L system (membrane expression of 
RANKL peaks around 48 hours and is present until 96 hours) and can be induced 
by various cytokines, such as IL-4 and transforming growth factor beta (TGFβ)38. 
RANKL can be membrane-bound on the surface of T cells, but also as a soluble 
receptor 39. Interestingly, serum levels of RANKL are increased in patients with 
various cancers 40. The receptor for RANKL, RANK, is physiologically expressed 
on the surface of various types of innate immunity cells, such as mature DC 41, 
natural killer (NK) cells 42, and monocytes/macrophages 43. In these immune cell 
subsets RANK signaling promotes survival and/or function. In the case of 


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osteoclasts, which are bone marrow-derived cells or MDSC, RANKL induces 
differentiation and bone loss. 
 
A recent study in syngeneic melanoma mouse models showed that the superior 
antitumor activity of concurrent CTLA-4 and RANKL blockade was dependent on 
T-cell and NK cell activity 37. There is accumulating evidence that the RANKL-
RANK-OPG system also plays an even more important role in tumor immunology. 
Among T cells that express RANKL, Tregs (CD4+Foxp3+) cells are the most 
interesting, because it has been shown that RANKL expression by Tregs may 
promote metastasis in RANK+ breast cancer cells 44. The influence of the RANKL-
RANK-OPG system on immune cell subsets that play an immunomodulatory role 
in cancers, such as MDSC and the alternatively activated macrophages (M2), is 
unknown. 
 
In the case of melanoma, both receptors for RANKL, RANK and OPG, were 
overexpressed by melanoma cells whereas RANKL was not. More specifically, 
RANK is expressed by melanoma-initiating cells (CD133+ or ABCB5+) and 
metastatic melanoma, implying that RANK may be involved in the development 
and maintenance of melanoma 45. OPG was upregulated by metastatic melanomas 
in response to TNFα 42. Given that the cell survival effect is conferred by RANK 
signaling through anti-apoptotic proteins (e.g. bcl-2 and bcl-x) in DC 46, it is 
reasonable to speculate that similar RANKL-mediated receptor pathways exist for 
melanoma cell survival. Thus, blocking RANKL with anti-RANKL antibody may, 
in addition to its effect in central immune tolerance, potentially have both direct 
antitumor (e.g. melanoma cell death), and indirect-immunomodulatory effects, 
such as: 1) blocking the suppressive effects of Tregs and,  2) blocking survival 
signals in MDSC and alternatively activated macrophages (M2).  
1.7 
Function of Thymus in Relation to Aging and Cancer; Facts and 
Unanswered Questions 
The thymus is an extremely sensitive organ to factors/conditions that are frequently 
prevalent in cancer, in particular conditions related to cancer treatment, such as 
irradiation, corticosteroids (either stress-induced or exogenously administered), 
and chemotherapy. It appears that both T-cell progenitors (most double positive 
thymocytes) and TEC can be adversely affected. Nevertheless, thymus can be 
regenerated, based on studies following allogeneic stem cell transplantation. Aging 
also causes involution of the thymus, which leads to defects in innate and adaptive 
immunity as well as immune dysregulation that may lead to autoimmune diseases 
47. However, using signal-joint TREC as a measurement of thymic function, studies 
have shown that the thymus continues to generate T cells, albeit in much lower 
numbers 48. Interestingly, our meta-analysis on patients who have been treated in 
large 
randomized 
controlled 
trials 
using 
ICP 
inhibitors 
(ipilimumab, 
tremelimumab, nivolumab, and pembrolizumab) and were dichotomized into 
younger and older patients with an age cutoff of 65-70 years showed that the 
clinical benefit in terms of prolongation of OS was similar in both younger and 
older patients, when all trials testing ICP inhibitors were grouped together. 


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However, the subgroup of older patients who were treated in 4 trials testing PD-1 
antibody therapies did not have clinical benefit compared to younger patients, 
suggesting either defects in adaptive immunity or different melanoma biology 49. 
Targeting pathways that regulate thymic growth and enhance thymic recovery is an 
active area of research 47. However, the impact of cancer on thymic function has 
not been systematically investigated beyond a handful of preclinical models 
(reviewed in 50) and only a few clinical trials in solid tumors have assessed the 
impact of systemic treatments in thymic function 51. 
 
It is important to emphasize that all preclinical experiments that Dr. Su and 
colleagues have performed on RANKL inhibition in melanoma were generated in 
young syngeneic melanoma mouse models. The clinical relevance of RANKL 
inhibition in patients with metastatic melanoma, 50% of whom are > 50 years old 
is therefore unknown. In this clinical trial we not only aim to assess the efficacy of 
denosumab in combination with ICP, but we also aim to systematically investigate 
the effect of this treatment on the abundance of various T-cell populations in 
peripheral blood (e.g. bone marrow T-cell precursors, thymocytes, recent thymic 
emigrants) as surrogate markers of influx and efflux from the thumus. 
 
As summarized above, we have identified that RANKL-mediated central immune 
tolerance is clinically relevant in cutaneous melanoma, based on preclinical 
observations and clinical findings. Thus, we will investigate the pharmacodynamic 
and antitumor effects of anti-PD-1 therapy in combination with denosumab 
administered at 120 mg s.c. q4 weeks in subjects with distant metastatic melanoma. 
Our hypothesis is that denosumab-anti-PD-1 drug combination therapy will be safe, 
well tolerated and may lead to antitumor responses that are significantly higher than 
those observed with a single anti-PD-1 agent.  
1.8 
Investigational Treatment – Denosumab Alone and with an Anti-PD-1 Agent 
 
Denosumab  
Denosumab is a human IgG2 monoclonal antibody that binds to soluble RANKL 
and is indicated for prevention of skeletal complications from bone metastases of 
any solid tumor malignancy 52. The standard dose is 120 mg administered s.c. every 
4 weeks. Although denosumab is generally well tolerated, common side effects 
include nausea (31%), diarrhea (20%), electrolyte abnormalities (hypocalcemia 
18%, hypophosphatemia 32%), fatigue (45%), and dyspnea (21%).  Denosumab 
has also been associated with osteonecrosis of the jaw (1.8%) and atypical femur 
fractures [see denosumab package insert]. We will administer additional extra 
“loading” doses of denosumab on day 8 and day 22 in our trial, prior to the initiation 
of an anti-PD-1 agent, in order to assess the pharmacodynamic and 
immunomodulatory effects of denosumab alone on central immune tolerance as 
well as assess any potential direct antitumor effects of denosumab.  
 
Nivolumab  


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Nivolumab is a potent and fully humanized monoclonal antibody of the 
immunoglobulin IgG4 that directly blocks the interaction between PD-1 and its 
ligands, PD-L1 and PD-L2. This blockade enhances functional activity of the target 
lymphocytes to facilitate an antitumor immune response, leading to tumor 
regression and immune rejection of the tumor. PD-L1 is expressed on 
approximately 40–50% of melanomas and has limited expression otherwise in most 
visceral organs with the exception of respiratory epithelium and placental tissue 53. 
The FDA has approved nivolumab as an adjuvant treatment for patients with 
completely resected melanoma with lymph node involvement or metastatic disease, 
based on findings from the phase III CheckMate-238 trial. In this trial in patients 
with stage IIIB/C or IV melanoma, the most commonly reported treatment-related 
adverse events with nivolumab were fatigue (34.5%), diarrhea (24.3%), pruritus 
(23.2%), rash (19.9%), and nausea (15% vs 20.1%). The most common grade 3/4 
treatment-related adverse events were diarrhea (1.5%), rash (1.1%), and increased 
ALT (1.1%). More recently, the FDA approved nivolumab dosing 480mg every 4 
weeks on the basis of similar time-averaged concentration, 16^ lower trough levels 
and 45% higher peak levels at steady-state54. The concordance of dosing for both 
nivolumab and denosumab and the lack of data suggesting that pembrolizumab and 
nivolumab are significantly different in terms of side effect profile and/or efficacy 
prompted us to allow for use of nivolumab in combination with denosumab in our 
study. 
 
Pembrolizumab  
Pembrolizumab is FDA-approved for upfront treatment of unresectable stage III or 
distant metastatic melanoma (AJCC stage III/IV) 10. Pembrolizumab is an IgG4 
kappa immunoglobulin that blocks the interaction between PD-1 and PD-L1, which 
decreases proliferation of T cells and production of cytokines. The standard dose 
for melanoma is 200 mg every 3 weeks.  Side effects are immune-mediated and 
include pneumonitis (2%, 0.4% grade 3), colitis (2%, 1.2% grade 3-4), hepatitis 
(1%, 0.8% grade 3-4), hypophysitis (0.8%, 0.4% grade 3-4), hyperthyroidism 
(3.3%, 0.1% grade 3), and hypothyroidism (8.1%, 0.1% grade 3) [see 
pembrolizumab package insert]. 
 
 
Combination of Denosumab and anti-PD1 Agents 
We do not feel that a phase 1 study of the denosumab-anti-PD-1 agent combination 
is required for two reasons: first, denosumab is usually well tolerated and, second, 
a significant number of patients with metastatic melanoma to the bone are already 
receiving  denosumab concomitantly with anti PD-1 therapy without reported 
incidents in peer-reviewed literature per standard of care. However, use of 
denosumab in combination with PD-1 inhibitors administered to a broader group 
of patients with metastatic melanoma, irrespective of bone metastases, has not been 
systematically investigated, short of retrospective case series 55,56. Admittedly, 
there is at least a theoretical premise for increased risk of colitis with the 
combination, based on preclinical experimental mouse models of colitis. In these 
models it was shown that the RANKL-RANK system is one of the important 
mediators of colitis. More specifically, RANKL was found to be expressed in 


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mucosal Treg cells and in subepithelial stromal cells within the domes of Peyer’s 
patches whereas RANK was expressed on CD11c+ (presumably dendritic) cells in 
the lamina propria (57; 58). In fact, inhibition of RANKL may result in a reduction 
of Treg cells in inflamed mucosa, and thus may worsen colitis 58. However, Dr. Su 
and colleagues have shown that Aire deficiency does not exacerbate colitis severity 
in mice that have been treated with CTLA-4 blocking antibody13. 
1.9 
Rationale for the LCCC1620 Study Design 
There is currently an early indication that denosumab may have effects beyond its 
well-described osteo-immunologic mechanism in cancer. For example, 
retrospective analysis of 811 patients with non-small cell lung cancer who have 
received denosumab versus zolendronic acid showed that denosumab prolonged 
median OS [8.9 months versus 7.7 months, hazard ratio (HR) 0.80, 95%CI 0.67-
0.95, p=0.01] 59. A retrospective analysis of patients with metastatic melanoma who 
had bone metastases, and therefore received denosumab in combination with 
immune checkpoint inhibitor(s), showed a trend towards longer OS in patients who 
received immune checkpoint inhibitor(s) plus denosumab, in particular for patients 
with visceral metastases (M1c) 56. RANKL was found to be a key paracrine effector 
of progesterone signaling in a subset of BRCA1 mutation carriers. In fact, 
denosumab treatment of patients with premenopausal BRCA1 mutation carriers 
resulted in substantial reduction of Ki67 staining seen in histologically normal 
tissue that was collected during the luteal phase 60, suggesting that denosumab may 
have chemopreventative effects in premenopausal RANK+ BRCA1-mutation 
carriers. 
1.9.1 Why Denosumab is Combined with PD-1 Blockade and Not CTLA-4 
Blockade? 
Dr. Su and colleagues have demonstrated that RANKL blockade in combination 
with CTLA-4 inhibition leads to higher anti-tumor responses via a mechanism that 
involves increased melanoma antigen-specific, tumor-infiltrating T cells (both 
CD4+ and CD8+), and, in particular, activated cytotoxic effector CD4+ cells 
(Figure 3 and Figure 4). While to date there is preclinical and clinical evidence 
(anecdotal case reports, 37) that RANKL blockade synergizes with CTLA-4 
blockade, there is only a single retrospective single-institution analysis of patients 
who received denosumab in combination with PD-1 and/or CTLA-4 inhibition56. 
Dr. Su and colleagues have shown in syngeneic B16-bearing mice that addition of 
RANKL inhibition to the combination with concurrent CTLA-4 and PD-1 
inhibition is more effective that CTLA-4/PD-1 inhibitor blockade alone (Figure 
3B). Furthermore, RANKL expression identifies tumor-infiltrating CD4+ and 
CD8+ T cells that expressing higher levels of PD-1 which can be modulated by 
anti-PD-161. In addition, the target, PD-1, is highly expressed in preclinical models 
of Aire deficiency (Figure 4). An additional, perhaps more important, reason is a 
clinical one; randomized phase 3 studies have consistently shown that PD-1 
blockade is far superior compared to CTLA-4 blockade 10. A triple combination 
(concurrent CTLA-4, PD-1, and RANKL blockade) may potentially be more 
effective than dual blockade (PD-1 and RANKL); however unforeseen side effects 


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may be observed with the triplet combination that are worse than ipilimumab-
nivolumab, which is already highly toxic. It is best to determine at this time if 
denosumab is safe and effective in combination with anti-PD-1 agent first before 
exploring the triplet combination, which may require a dedicated phase I study. 
1.9.2 Why Three “Loading” Doses of Denosumab Prior to Anti-PD-1 Agent 
Administration? 
Dr. Su and colleagues have shown that in order for concurrent RANKL and CTLA-
4 blockade to be effective, RANKL blockade must precede treatment with CTLA-
4 blockade 13. In addition, in the single-group, open-label, multicenter study of 
denosumab in patients with giant-cell tumor of the bone a total of three loading 
doses (day 1, day 8, and day 15) during the first 4 weeks was not associated with 
any new or worse side effects 62. Therefore, we believe that two additional loading 
doses of denosumab during the first 3 weeks will be safe, and would therefore 
mitigate the need for a dedicated phase I study.  
1.9.3 Primary Endpoints are Translational Investigations and Not Clinical 
Efficacy? 
While this study aims to investigate the clinical benefit of the denosumab-anti-PD-
1 agent combination, this is not the primary objective, and as such the study is 
underpowered for efficacy. Our study will investigate the pharmacodynamic effects 
of denosumab alone and in combination with an anti-PD-1 agent on central immune 
tolerance as well as denosumab’s direct effect on tumor tissues collected on day 22 
after three loading doses of denosumab are given on day 1, day 8 and day 22. As 
described in the Correlative Studies section (Section 1.10), we plan to perform 
analysis in peripheral blood and tumor tissue to investigate thymic input and output 
(see Figure 2) using multi-parameter flow cytometry and sjTREC analysis as well 
as the effect of the treatment combination in tumor tissue. The results from these 
three translational analyses will provide insights into the impact of RANKL 
inhibition and its role in abrogating central immune tolerance as well as highlight 
any potential direct effect RANKL inhibition may have on melanoma and the 
abrogation of peripheral immune tolerance. If there is a signal of clinical activity 
and benefit that is supported by correlative studies, future phase 1-3 studies may be 
conducted to combine denosumab with CTLA-4 and/or PD-1 inhibitors, and 
therefore expand the label of use of denosumab beyond the current one. 
1.10 
Correlative Studies 
The co-primary endpoints of this study require collection of PBMCs, serum 
samples, and tumor tissue biopsies to understand the immunomodulatory and 
potentially direct antitumor effects of denosumab alone and in combination with an 
anti-PD-1 agent. The ability to collect tumor tissues at baseline and after three 
weeks of denosumab monotherapy will allow us to investigate the 
immunomodulary role of denosumab-alone treatment. Anti-PD-1 therapy will be 
initiated after samples are collected on day 22 (at the start of week 4) to explore the 
effects of combination therapy. The ability to collect peripheral blood at baseline; 
after three weeks of denosumab and then at weeks 16, 28, and 40, during 


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combination therapy coupled with disease restaging studies will allow us to assess 
the early effects of denosumab alone versus the early and late effects of the 
combination of denosumab and an anti-PD-1 agent.  
1.10.1 Peripheral Blood 
1.10.1.1 Serum levels of RANKL (free and OPG-bound) and OPG 
These will be measured by ELISA (Su’s lab). We predict serum OPG will be low, 
serum-free RANKL will be considerably high whereas the serum OPG/RANKL 
ratio will be low in a significant number of patients with metastatic melanoma at 
baseline. In fact, we predict that patients with high serum-free RANKL and/or low 
serum OPG levels will receive the greatest benefit from anti-PD-1-denosumab 
combination therapy. Following treatment with denosumab, we project that free 
RANKL will significantly decrease. If denosumab administered at the FDA-
approved dose is able to bind free RANKL, then undetectable levels of free 
RANKL are expected. 
1.10.1.2 Peripheral Blood Mononuclear Cell Immune Monitoring 
The following cell populations will be ennumerated using multiparameter flow 
cytometry with the following decreasing order of importance: 
 
(1) Bone marrow-derived double negative early thymocytes (baseline, week 4, 
week 16, 28, and 40; Su’s lab) CD34+CD2+CD4-CD5+CD7+CD8-CD45RA+ 
(see Figure 2). We anticipate no changes following treatment with denosumab 
plus anti-PD-1 agent., although overall numbers may be significantly lower 
compared to same-age individuals who do not have cancer (i.e. patients’ 
relatives). 
 
(2) RTE by flow cytometric analysis (baseline, week 4, week 16, 28, and 40; 
Su’s 
lab) 
CD4+CD45RO-CD45RA+CD62L+CD31+, 
CD4+Foxp3+ 
CD45RO-CD45RA+CD62L+CD31+, 
and 
CD8+CD45RO-
CD45RA+CD62L+CD31+ (see Figure 2). We anticipate that denosumab 
alone will slightly increase total output of CD8+ and CD4+ non-Treg RTE, as 
well as CD4+ Tregs, although changes may be borderline. However, TCR 
repertoire analysis on fluorescence activated cell sorting (FACS)-sorted CD8+ 
RTE, CD4+ non-Tregs RTE, and CD4+ Tregs RTE will likely show increase 
of distinct clones for CD4+ non-Tregs and CD8+, and decrease of distinct 
clones for CD4+ Tregs. 
 
(3) RTE by sjTREC analysis on FACS-sorted CD4+ and CD8+ PBMCs 
(baseline, week 4, week 16, 28, and 40; Duke Human Vaccine Institute, Dr. 
Gregory D Sempowski). The reason why we propose two independent assays 
for assessment of RTE is because TRECs do not solely reflect recently 
emigrated cells from the thymus, but also long-lived naïve cells in the 
periphery. In fact, proliferation of naïve T-cells in the periphery is the 
dominant homeostatic mechanism of T cell numbers in the elderly48. 


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(4) Immune cell receptor repertoire analysis on FACS-sorted CD4+ and 
CD8+ PBMCs (baseline, week 4, week 16, 28, and 40; Immune Genomics 
Facility, UNC-CH, Dr. Benjamin Vincent). Treatment with PD-1 inhibitors 
alone leads to significant increases in the size of distinct immune receptor 
clones (both TCR and B-cell receptor) without changes in diversity 63. We 
postulate that treatment with denosumab alone will increase thymic output of 
autoreactive clones, and therefore will increase clonal diversity. When 
denosumab is co-administered with an anti-PD-1 agent, we will observe a 
combination of increase size in diversity as well as clonality. 
 
(5) Effector T cell subtypes (baseline, week 4, week 16, 28, and 40; Su’s lab), 
such as cytotoxic (CD3+CD4-CD8+Granzyme B+) and exhausted 
(CD3+CD4-CD8+PD1+TIM3+LAG3+). RANKL is upregulated on activated 
T cells within 48 hours and are sustained until 96 hours 38. There is little 
knowledge about the effect of RANKL inhibition in effector T cells in cancer, 
which may well bear the exhausted phenotype. We anticipate that denosumab 
alone will not affect numbers of activated effector T cells, whereas the effect 
on exhausted phenotype T cells is unknown, but important to explore. 
  
(6) Treg cells (baseline, week 4, week 16, 28, and 40; Su’s lab) 
CD4+CD25high+FoxP3+(RANKL) 
Assuming that RANKL transmits a survival effect on RANK+ Tregs 44, 
treatment with denosumab alone will decrease the number of RANK+ Tregs. 
Pembrolizumab did not increase the number of intratumoral Tregs 64. 
Therefore, we do not anticipate further reduction of peripheral Tregs, including 
RANK+ ones, when PD-1 inhibitors are concurrently administered with 
denosumab. 
 
(7) MDSC (baseline, week 4, week 16, 28, and 40; Su’s lab) monocytic (HLA-
DRlowCD14+), 
other 
monocytic 
(lin1—HLA-DR—CD33+CD11b+), 
lymphoid (lin1—HLA-DR—CD33+CD11b+) 
We cannot predict the outcome of distinct MDSC numbers and types when 
denosumab will be administered in combination with an anti-PD-1 agent, but 
the result will be important to explore. It is possible that RANKL transmits 
survival signals to alternatively activated MDSC, and therefore RANKL 
inhibition will suppress distinct subtypes of MDSC. Pembrolizumab increased 
number of intratumoral monocytic MDSC 64. Suppression of all MDSC by 
denosumab in combination with anti-PD-1 agent will be beneficial. 
1.10.2 Tumor Tissue 
The goal of tumor tissue collection is to investigate the effect of denosumab-alone 
in tumor biopsies, both in terms of mechanistic (immunomodulatory and/or direct-
antitumor) as well as pharmacodynamic effects (i.e. saturation of the denosumab 
target within tumor tissue, as we have previously shown with other antibody 
therapies in melanoma65). 


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Optional archived tumor tissue will be collected from any procdures that took place 
prior to study enrollment. Biopsies on day 22 will be mandatory because 
investigation of denosumab-alone effect in tumor tissue is a co-primary endpoint. 
As a co-primary aim, we would like to perform tumor biopsies at week 16 (i.e. 12 
weeks after both anti-PD-1 agent and denosumab are concurrently administered) to 
assess the combined effect of these agents. Tumor biopsies at the end of week 16 
will be optional, because we believe that a second mandatory biopsy within a 
clinical trial may impair subject accrual. Optional archived tumor tissue will also 
be collected on any procedures that occur after study completion or study removal 
due to tumor progression. Given the small size of excisional biopsies, it is 
impossible to perform flow cytometry to assess melanoma-infiltrating immune cell 
subsets on these samples. Rather, excised tumors will be fixed in formalin and 
paraffin-embedded (FFPE). Hematoxylin and eosin (H&E)-stained tissue sections 
will be generated to assess the quality (necrosis) and amount of tumor cells present. 
In addition, the amount of TILs will be scored, as previously described 33. 
Furthermore, 5-µm thick tissue sections will be prepared to perform tumor-imaging 
analysis (all in Translational Pathology Laboratory, TPL, University of North 
Carolina at Chapel Hill, UNC-CH) in the following decreasing level of importance. 
 
1.10.2.1 Immunohistochemistry; single-stain (baseline, week 4, and/or week 16) 
(1) CD8 stain; we anticipate that denosumab alone will increase tumor-
infiltrating CD8+ cells (baseline →day 22), as Dr. Su has shown previously 
in Aire-deficient syngeneic melanoma-bearing mouse models 30. This 
increase will be further augmented when anti-PD-1 agent is added 64. 
(2) RIP3 and Apoptag stain. The reason for this analysis is because both 
RANK and OPG are expressed by melanoma cells and transmit survival 
signals 42,45. We postulate that denosumab alone will increase cell death 
(necroptosis and/or apoptosis).  
(3) RANKL stain using denosumab as a primary antibody. To investigate 
whether denosumab administered at 120 mg s.c. on day 1 and day 8 
sufficiently binds (“saturates”) all RANKL sites on the cell membrane of 
cells within tumor tissue (presumably Treg, other T cells) or stromal cells, 
we will use denosumab as a primary antibody for IHC. Tissue saturation 
studies of the antibody target in tumor tissue have been previously used to 
assess whether the dose/schedule of antibody therapy is adequate65. We 
anticipate that if denosumab fully saturates tissue-associated RANKL, no 
IHC stain will be observed on day 22 compared to baseline, similar to our 
previously published pharmacodynamic studies using monoclonal antibody 
therapies 65. 
1.10.2.2  3-color IF (baseline) 
(1) Sox10 (melanoma-specific transcription factor), RANK, OPG (baseline 
only). We would like to investigate expression of receptors for RANKL in 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
28 
melanoma cells. We postulate that expression of any of the two RANKL 
receptors correlates with clinical benefit from denosumab-anti-PD-1 agent. 
(2) CD8, RANKL, PD1 (baseline only). We would like to investigate 
expression of RANKL in total CD8+ and exhausted (PD-1+) effector T cells 
to assess degree of colocalization of RANKL and PD-1 in immune cells at 
baseline.  
(3) CD68, CD163 (baseline, day 22, and/or week 16). RANK is known to 
be expressed in macrophages 43. The role of macrophages in cancer is 
controversial, but there are reports suggesting a tumor-promoting role. We 
postulate that denosumab will disrupt RANK-dependent survival signals in 
tumor-associated macrophages (TAM). Therefore, we anticipate that the 
number of TAM will decrease on day 22 and, even more so, on week 16. 
(4) CD33, CD11b, HLA-DR (baseline, day 22, week 16). RANK is also 
expressed in MDSC 46, which are precursors for osteoclast progenitors. 
MDSC may suppress effector T-cell functions. We postulate that, similar to 
TAM, RANK transmits survival signals to MDSC. We therefore postulate 
the number of tumor-infiltrating MDSC will decrease on day 22 and, even 
more so, at week 16.  


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CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
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 July 7, 2022 
29 
2.0 
STUDY OBJECTIVES 
2.1 
Co-primary Objectives 
2.1.1 Assess the mechanistic (immune-mediated and/or direct antitumor effect) and 
pharmacodynamics effect (tissue saturation studies) of denosumab alone (i.e., after 
three loading doses of denosumab are given on day 1, 8 and day 22) in patients with 
unresectable (or resectable) stage III or distant metastatic PD-1/PD-L1 inhibitor-
naïve* (*, see exceptions to this rule in sections 3.1 and 3.2 Inclusion/Exclusion 
criteria) melanoma (stage III/IV) by performing translational research on peripheral 
blood and tumor biopsy samples collected at baseline and after third loading dose 
of denosumab. 
2.1.2 Assess the immune-mediated and direct antitumor effect of denosumab in 
combination with anti-PD-1 agent in patients with unresectable (or resectable) stage 
III or distant metastatic PD-1/PD-L1 inhibitor-naïve* (*, see exceptions to this rule 
in sections 3.1 and 3.2 Inclusion/Exclusion criteria) melanoma (AJCC stage III/IV) 
by performing translational research on peripheral blood and tumor biopsy samples 
collected at weeks 16, 28 and 40 of the study and comparing the results with those 
from baseline and after third loading dose of denosumab. 
 
2.2 
Secondary Objectives  
2.2.1 Assess the safety of the denosumab-anti-PD-1 agent combination in unresectable 
(unresectable) stage III or distant metastatic PD-1/PD-L1 inhibitor-naïve* (*, see 
exceptions to this rule in sections 3.1 and 3.2 Inclusion/Exclusion criteria) 
melanoma (AJCC stage III/IV) by NCI-CTCAE version 5.0. 
2.2.2 Determine antitumor response by RECIST v1.1 criteria of the denosumab-anti-PD-
1 agent combination at 16 weeks in patients with unresectable (resectable) stage III 
or distant metastatic PD-1/PD-L1 inhibitor-naïve* (*, see exceptions to this rule in 
sections 3.1 and 3.2 Inclusion/Exclusion criteria) melanoma (AJCC stage III/IV). 
2.2.3 Determine the 1-year OS rate of the denosumab-anti-PD-1 agent combination in 
patients with unresectable (or resectable) stage III or distant metastatic PD-1/PD-
L1 inhibitor-naïve* (*, see exceptions to this rule in sections 3.1 and 3.2 
Inclusion/Exclusion criteria) melanoma (AJCC stage III/IV). 
2.2.4 Determine the 6-month PFS rate of the denosumab-anti-PD-1 agent combination in 
patients with unresectable (or resectable) stage III or distant metastatic PD-1/PD-
L1 inhibitor-naïve* (*, see exceptions to this rule in sections 3.1 and 3.2 
Inclusion/Exclusion criteria) melanoma (AJCC stage III/IV). 
2.3 
Exploratory Objectives  
 










LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
34 
Note: Abstinence is acceptable if this is the usual lifestyle and preferred contraception 
for the subject. 
4.1.1.13 As determined by the enrolling physician or protocol designee, willingness and ability of 
the subject to understand and comply with study procedures. 
4.1.1.14 Previous radiation therapy is allowed, provided it is completed ≥14 days prior to starting 
therapy and subject has recovered adequately from any related toxicities (grade ≤1, or 
grade ≤2 that is stable for ≥3 months). 
4.1.1.15 If subject has received adjuvant treatments, in particular ipilimumab and high dose 
interferon, any toxicities must have resolved to grade 1 or less. Grade 2 toxicities 
attributed to ipilimumab from autoimmune endocrinopathies that require permanent 
hormone replacement therapy are allowed as long as they are adequately treated. This 
implies that subjects should be off systemic steroids for treatment of any of these or other 
autoimmune toxicities (e.g. colitis, rash). 
4.1.1.16 Subjects who have previously received PD-1 inhibitors in stage III (adjuvant) or stage 
IV are allowed as long as:  
(a) the interval between the last dose of the adjuvant PD-1 inhibitor and the date of 
relapse (clinical or radiographic) is at least 1 year,  
(b) if subjects who received treatment for stage IV had antitumor response (partial 
response or complete response) by RECIST criteria version 1.1 but they stopped 
due to subject/investigator preference for at least a year between the last dose of the 
PD-1 inhibitor and the date of relapse (clinical or radiographic). Allowing for these 
subjects who have previously received PD-1 inhibitors in the adjuvant setting (i.e. 
no knowledge about clinical benefit) or following definite antitumor response in 
the metastatic setting is based on a recent case series of subjects who responded to 
PD-1 inhibitor rechallenge, if they had previously responded to PD-1 inhibitors67. 
This implies that waning antitumor immunity in the absence (i.e. >1 year) of 
costimulation with PD-1 inhibitors may be the reason for cancer recurrence and 
NOT primary resistance of PD-1 inhibitors. 
(c) any side effects that may have occurred during the previous exposure to PD-1 
inhibitors are not serious (i.e. grade 1 or 2 by CTCAE version 5.0 criteria). 
 
 
4.2 
Exclusion Criteria 
4.2.1.1 History of prior malignancy, with the exception of the following: 
• Non-melanoma skin cancers, non-invasive bladder cancer, and carcinoma in situ of the 
cervix, 
• Prior history of prostate, provided that subject is not under active systemic treatment 
other than hormonal therapy and with documented undetectable prostate specific 
antigen (PSA < 0.2 ng/mL), 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
35 
• Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) provided 
subject has isolated lymphocytosis (Rai stage O), and does not require systemic 
treatment [for “B” symptoms, Richter’s transformation, lymphocyte doubling time (< 
6 months), lymphadenopathy or hepatosplenomegaly], 
• Lymphoma or any type of hairy-cell leukemia, provided subject is not on an active 
systemic treatment and is in complete remission, as evidenced by PET/CT scans and 
bone marrow biopsies for at least 3 months, 
• History of other malignancy, provided subject has completed therapy, or does not 
require therapy, and is free of disease for ≥ 2 years. If subject has had other malignancy 
within the last 2 years from which he/she may have been completely cured by surgery 
alone, or does not require any treatment other than observation at the specialist’s 
discretion, he/she may considered to be enrolled on condition that the risk of 
development of recurrent or distant metastatic disease based on the American Joint 
Committee in Cancer (AJCC) staging system is less than 30% in 3 years from the 
original diagnosis of other malignancy. 
4.2.1.2 Has known active central nervous system (CNS) metastases that are symptomatic and 
require antiepileptic drugs or corticosteroids (any dose). Subjects with previously treated 
brain metastases may participate provided they are asymptomatic (i.e., no neurologic 
symptoms) for at least 2 weeks prior to the first dose of trial treatment and are not using 
steroids for at least 7 days prior to trial treatment. Patients with previously treated brain 
metastases should have evidence of stable brain metastases (without evidence of 
progression by imaging) for at least 2 weeks prior to the first dose of trial treatment. 
Patients with previously treated but grown brain metastases (or new brain metastases if 
there is no prior history of brain metastases) are still allowed in the study as long as the 
net growth of pre-existing brain lesions (or new brain lesions if there is no prior history 
of brain metastases) does not exceed 1-cm in largest diameter as measured by brain MRI 
with IV contrast (for example, a patient with pre-existing brain lesion(s) that has grown 
by 6-mm in largest diameter and also has a new lesion that measures 4-mm in largest 
diameter can still be enrolled in the study; alternatively, a patient that has a new brain 
lesion that measures 1-cm in largest diameter and no growth of pre-existing brain lesions 
can still be enrolled in the study). This exception does not include carcinomatous 
meningitis, which is excluded regardless of clinical stability. Subjects with 
leptomeningeal disease, detected either by brain MRI or by cytology (e.g. lumbar 
puncture) are also excluded.  
4.2.1.3 Treatment with any investigational drug, immunotherapy or chemotherapy within 28 
days prior to study treatment (i.e., initiation of denosumab). Treatment with any targeted 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
36 
therapy (e.g. MAPK inhibitors) is allowed as long as at least 15 days have elapsed since 
last dose of drug. 
4.2.1.4 Subjects discontinuing prior therapy with tyrosine kinase inhibitors for melanoma should 
be off these medications for at least 15 days before starting study treatment. 
4.2.1.5 Prior PD-1/PD-L1 therapies in the adjuvant setting (with exceptions noted in inclusion 
Section 3.1.1.16); targeted therapies or prior ipilimumab in the adjuvant setting are 
allowed.  
4.2.1.6 Any condition, including laboratory abnormalities, that in the opinion of the investigator 
places the subject at unacceptable risk, if he/she were to participate in the study.  This 
includes, but is not limited, to serious medical conditions or psychiatric illness likely to 
interfere with participation in this clinical study. 
4.2.1.7 Has a diagnosis of immunodeficiency or is receiving systemic steroid therapy equivalent 
to daily doses of prednisone of 10 mg or greater (or an equivalent dose of other 
corticosteroids) or any other form of immunosuppressive therapy within 7 days prior to 
the first dose of trial treatment. 
4.2.1.8 Has a known history of active tuberculosis (Mycobacterium Tuberculosis).  
4.2.1.9 Is pregnant or breastfeeding, or expecting to conceive or father children within the 
projected duration of the trial, starting with the pre-screening or screening visit through 
120 days after the last dose of trial treatment. 
4.2.1.10 Hypersensitivity to nivolumab, pembrolizumab or denosumab or any of their excipients. 
4.2.1.11 Has active autoimmune disease that has required systemic treatment in the past 1 year 
(i.e. with use of disease-modifying agents, corticosteroids or immunosuppressive drugs). 
Replacement therapy (e.g., thyroxine, insulin, or physiologic corticosteroid replacement 
therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic 
treatment.  
4.2.1.12 Has a history of non-infectious pneumonitis that required systemic corticosteroids or 
evidence of interstitial lung disease or current active, non-infectious pneumonitis. 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
37 
Episodic, brief (< 7 day) exposure to systemic corticosteroids (e.g. steroid taper for 
poison ivy or COPD exacerbation) is allowed. 
4.2.1.13 Has a history of an acute coronary event (e.g. myocardial infarction) within 3 months 
since study entry, uncontrolled and symptomatic coronary artery disease, or congestive 
heart failure New York Heart Association class III/IV. 
4.2.1.14 Has an active infection requiring systemic therapy within 7 days prior to treatment 
initiation. 
4.2.1.15 Has a known history of Human Immunodeficiency Virus (HIV 1/2 antibodies). 
4.2.1.16 Known serologic status reflecting active hepatitis B or C infection. Subjects that are 
hepatitis B core antibody positive, but antigen negative, will need a negative polymerase 
chain reaction (PCR) prior to enrollment.  [NOTE: Hepatitis B antigen or PCR positive 
subjects will be excluded]. 
4.2.1.17 Has received a live vaccine within 30 days of planned start of study therapy. NOTE: 
Seasonal influenza vaccines for injection are generally inactivated flu vaccines and are 
allowed; however intranasal influenza vaccines (e.g., Flu-Mist®) are live attenuated 
vaccines, and are not allowed. 
4.2.1.18 Known active metabolic bone disease such as Paget’s disease, Cushing’s disease, 
hyperprolactinemia, over the last year 12 months, known history of osteoporosis that is 
symptomatic (e.g. history of fractures, bone pain), or hypercalcemia/hypocalcemia of any 
type (serum free calcium being more than 1.1 × ULN and less than 0.9 x, lower limits 
normal. LLN) over the last 2 weeks since study initiation that requires treatment beyond 
calcium and vitamin D supplementation.  
4.2.1.19 Prior treatment with denosumab. Use of bisphosphonates for treatment of metastatic bone 
disease, but not for hypercalcemia of malignancy, is allowed. 
4.2.1.20 History of current evidence of osteonecrosis or osteomyelitis of the jaw. Note: Subject 
should be referred to dentist before study treatment initiation for poor dentition or other 
dental issues that, in the opinion of the treating physician, may increase the risk of 
osteonecrosis of the jaw. 
 
 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
38 
5.0 
TREATMENT PLAN 
5.1 
Schema 
 
 
Figure 6. LCCC1620 Study Schema. Abbreviations: SC, subcutaneous; D, day; Wk, week; RECIST, 
Response Evaluation Criteria in Solid Tumors; OS, overall survival; Tx, treatment; AE, adverse events; pt, 
patient; PBMC, peripheral blood mononuclear cells; Tregs, T regulatory cells; MDSC, myeloid-derived 
suppressor cells; TCR, T-cell receptor; OPG, osteoprotegerin; IHC, immunohistochemistry; IF, 
immunofluorescence. 
 
Treatment schema is shown in Figure 6. Eligible subjects must have AJCC stage III/IV 
melanoma of cutaneous or mucosal (or unknown) primary without any prior treatment with 
a PD-1 inhibitor or denosumab and available archived tumor blocks from prior melanoma 
surgeries. Please see Section 3.1.1.16 for exceptions to the PD-1 inhibitor naïve rule. 
Alternatively, if subjects do not have tumor tissue blocks from metastatic melanoma, they 
can be enrolled into the study if they undergo an excisional or CT-guided biopsy. At 
baseline, peripheral blood and archived (or fresh) tumor blocks will be collected for 
correlative studies. Leftover or excess research tumor tissue may be collected during all 
standard of care surgical procedures prior to enrollment, during study participation and will 
continue during follow up until death. To investigate denosumab-induced changes in 
peripheral blood and tumor tissue, denosumab will be administered first on day 1, day 8 
and day 22 (day 29 prior to Amendment 1) and concomitant anti-PD-1 agent will be 
initiated on day 22. Immediately prior to the first infusion of anti-PD-1 agent (nivolumab 
or pembrolizumab), peripheral blood and a mandatory tumor biopsy will be collected. 
Following the day 22 time point, pembrolizumab will be administered every 3 weeks (in 
subjects enrolled prior to Amendment 1), nivolumab (in subjects enrolled after Amendment 
1) and denosumab will be administered every 4 weeks as combination therapy for up to 1 
year. Restaging radiographic studies will be performed at week 16 and then every 12 weeks 
thereafter, as per standard of care; peripheral blood will be collected at the same time points 
and an optional biopsy will be performed at week 16 to investigate denosumab plus anti-


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
39 
PD-1 therapy effects on tumor tissue. Subjects will be considered to have completed all 
trial requirements if: 
(a) their disease has progressed by RECIST version 1.1 criteria within less than a year 
from study initiation, 
(b) they develop toxicity related to any of the study drugs, but still complete sample 
collection (peripheral blood and tumor tissue) and tumor assessments by at least week 
16, 
(c) complete the entire year of combination therapy and study-related assessments 
without experiencing disease progression. Those subjects experiencing clinical benefit 
after 1 year may continue anti-PD-1 therapy per standard of care. 
5.2 
Treatment Dosage and Administration 
Subjects in this trial will be given denosumab, 120 mg s.c. every 4 weeks, starting on day 
1 of study treatment. Additional loading doses of denosumab will be administered on day 
8 and day 22 (after Amendment 1). Nivolumab, 480 mg will be administered intravenously 
(IV) every 4 weeks and initiated 21 days after the first dose of denosumab is given. In 
subjects enrolled prior to Amendment 1 pembrolizumab, 200 mg will be administered 
intravenously (IV) every 3 weeks and initiated 21 days after the first dose of denosumab is 
given. Combination therapy with both agents will continue as long as subjects benefit from 
therapy for up to 1 year. Study therapy will be discontinued for intolerable toxicity, disease 
progression or for other reasons at the discretion of the investigator. If subjects are not 
withdrawn prematurely then their last dose of study medications will be administered 
approximately 49 weeks after denosumab was initiated.   
 
 
Agent 
Required Premedications; 
Precautions 
 
Dose 
 
Route 
 
Schedule 
Denosumab 
Correct hypocalcemia prior to 
initiating denosumab therapy. 
Monitor calcium levels during 
therapy, especially during the 
first 8 weeks of therapy, and 
adequately supplement all 
subjects with calcium (≥ 500mg 
qd) and vitamin D (≥ 400 IU 
qd). Monitor calcium, 
magnesium and phosphorus. 
120 mg 
s.c.; 
Administer in 
upper arm, 
upper thigh or 
abdomen 
Every 28 
days.  
 
Additional 
loading 
doses of 
120mg s.c. 
will be 
administered 
on days 8 
and 22 
Nivolumab 
Risk for immune-mediated and, 
infusion-related reactions  
 
480 mg 
IV; 
Administer 
over 30 min 
Every 28 
days 
Pembrolizumab 
Risk for immune-mediated1 and, 
infusion-related2 reactions  
 
200 mg3 
 
IV; 
Administer 
over 30 min  
Every 21 
days 
1. See Section 4.3.4 for management guidelines for immune-mediated AEs 
2. See Section 4.5.1 for management guidelines for infusion-related AEs 
3. 
Note: On days when denosumab is administered on the same day as anti-PD-1 agent, the s.c. injection 
should be given after the infusion of the anti-PD-1 agent is completed. 
 








LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
43 
5.3.3 Management of Nivolumab Infusion Reactions 
Nivolumab can cause severe infusion reactions which have been reported in less than 1.0% 
of subjects.  If such a reaction were to occur, symptoms would manifest as fever, chills, 
rigors, headache, rash, pruritis, arthralgias, hypo- or hypertension, bronchospasm, or other 
symptoms of allergic-like reactions.  
 
All Grade 3 or 4 infusion reactions should be reported as an SAE if criteria are met. Infusion 
reactions should be graded according to NCI CTCAE v5.0 guidelines. 
 
Treatment recommendations are provided below and may be modified based on local 
treatment standards and guidelines as appropriate. 
 
For Grade 1 symptoms during nivolumab infusion (mild reaction; infusion interruption 
not indicated; intervention not indicated). Remain at bedside and monitor subject until 
recovery from symptoms. The following prophylactic pre-medications are recommended 
for future infusions: diphenhydramine 50 mg (or equivalent) and/or paracetamol 325 to 
1000 mg (acetaminophen), at least 30 minutes before additional nivolumab 
administrations. 
 
For Grade 2 symptoms during nivolumab infusion (moderate reaction requires therapy 
or infusion interruption but responds promptly to symptomatic treatment (e.g., 
antihistamines, non-steroidal anti- inflammatory drugs, narcotics, corticosteroids, 
bronchodilators, IV fluids); prophylactic medications indicated for 24 hours).  
 
Stop the nivolumab infusion, begin an IV infusion of normal saline, and treat the subject 
with diphenhydramine 50 mg IV (or equivalent) and/or paracetamol 325 to 1000 mg 
(acetaminophen); remain at bedside and monitor subject until resolution of symptoms. 
Corticosteroid or bronchodilator therapy may also be administered as appropriate. If the 
infusion is interrupted, then restart the infusion at 50% of the original infusion rate when 
symptoms resolve; if no further complications ensue after 30 minutes, the rate may be 
increased to 100% of the original infusion rate. Monitor subject closely. If symptoms recur, 
then no further nivolumab will be administered at that visit. Administer diphenhydramine 
50 mg IV, remain at bedside, and monitor the subject until resolution of symptoms. The 
amount of study drug infused must be recorded on the electronic case report form (eCRF). 
The following prophylactic pre-medications are recommended for future infusions: 
diphenhydramine 50 mg (or equivalent) and/or paracetamol 325 to 1000 mg 
(acetaminophen) should be administered at least 30 minutes before additional nivolumab 
administrations. If necessary, corticosteroids (recommended dose: up to 25 mg of IV 
hydrocortisone or equivalent) may be used. 
 
For Grade 3 or Grade 4 symptoms during nivolumab infusion: (Severe reaction, Grade 
3: prolonged [i.e., not rapidly responsive to symptomatic medication and/or brief 
interruption of infusion]; recurrence of symptoms following initial improvement; 
hospitalization indicated for other clinical sequelae [e.g., renal impairment, pulmonary 
infiltrates]). Grade 4: (life threatening; vasoactive agents for hemodynamic support or 
mechanical ventilation indicated).  




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CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
45 
• 
Add prophylactic 
antibiotics for 
opportunistic infections 
Diarrhea 
/ Colitis  
Grade 2 or 3 
Withhold 
• Administer 
corticosteroids (initial 
dose of 1-2 mg/kg 
prednisone or 
equivalent) followed by 
taper 
 
• 
Monitor participants for 
signs and symptoms of 
enterocolitis (i.e., 
diarrhea, abdominal pain, 
blood or mucus in stool 
with or without fever) 
and of bowel perforation 
(ie, peritoneal signs and 
ileus). 
• 
Participants with ≥ Grade 
2 diarrhea suspecting 
colitis should consider 
GI consultation and 
performing endoscopy to 
rule out colitis.  
• 
Participants with 
diarrhea/colitis should be 
advised to drink liberal 
quantities of clear fluids.  
If sufficient oral fluid 
intake is not feasible, 
fluid and electrolytes 
should be substituted via 
IV infusion. 
Grade 4 
Permanently 
discontinue 
AST / 
ALT 
elevation 
or 
Increased 
bilirubin 
Grade 2 
Withhold 
• Administer 
corticosteroids (initial 
dose of 0.5- 1 mg/kg 
prednisone or 
equivalent) followed by 
taper 
• 
Monitor with liver 
function tests (consider 
weekly or more 
frequently until liver 
enzyme value returned to 
baseline or is stable 
Grade 3 or 4  
Permanently 
discontinue 
• Administer 
corticosteroids (initial 
dose of 1-2 mg/kg 
prednisone or 
equivalent) followed by 
taper 
Type 1 
diabetes 
mellitus 
(T1DM) 
or 
Hypergly
cemia 
 
Newly onset 
T1DM or  
Grade 3 or 4 
hyperglycemia 
associated with 
evidence of β-cell 
failure 
Withhold 
• Initiate insulin 
replacement therapy for 
participants with T1DM  
• Administer anti-
hyperglycemic in 
participants with 
hyperglycemia  
 
• 
Monitor participants for 
hyperglycemia or other 
signs and symptoms of 
diabetes. 
Hypophy
sitis 
Grade 2 
Withhold 
• Administer 
corticosteroids and 
initiate hormonal 
replacements as 
clinically indicated.   
 
• 
Monitor for signs and 
symptoms of 
hypophysitis (including 
hypopituitarism and 
adrenal insufficiency)  
Grade 3 or 4  
Withhold or 
permanently 
discontinue1  


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
46 
Hyperthy
roidism  
Grade 2 
Continue  
• Treat with non-selective 
beta-blockers (e.g., 
propranolol) or 
thionamides as 
appropriate  
• 
Monitor for signs and 
symptoms of thyroid 
disorders.  
 
Grade 3 or 4  
Withhold or  
permanently 
discontinue1 
Hypothyr
oidism 
Grade 2-4 
Continue  
• Initiate thyroid 
replacement hormones 
(e.g., levothyroxine or 
liothyroinine) per 
standard of care 
• 
Monitor for signs and 
symptoms of thyroid 
disorders.  
 
Nephritis 
and 
Renal 
dysfuncti
on 
Grade 2 
Withhold  
• Administer 
corticosteroids 
(prednisone 1-2 mg/kg 
or equivalent) followed 
by taper. 
• 
Monitor changes of renal 
function 
 
Grade 3 or 4  
Permanently 
discontinue 
Myocardi
tis 
Grade 1 or 2 
Withhold 
• Based on severity of 
AE administer 
corticosteroids 
• 
Ensure adequate 
evaluation to confirm 
etiology and/or exclude 
other causes 
 
Grade 3 or 4 
Permanently 
discontinue 
All other 
immune-
related 
AEs 
Intolerable/ 
persistent Grade 2 
Withhold 
• Based on type and 
severity of AE 
administer 
corticosteroids  
• 
Ensure adequate 
evaluation to confirm 
etiology and/or exclude 
other causes 
 
Grade 3 
Withhold or 
discontinue 
based on the 
type of event.  
Events that 
require 
discontinuation 
include and not 
limited to:  
Guillain-Barre 
Syndrome, 
encephalitis 
Grade 4 or 
recurrent Grade 3  
Permanently 
discontinue 
1. Withhold or permanently discontinue pembrolizumab is at the discretion of the investigator or treating 
physician.   
NOTE:  
For participants with Grade 3 or 4 immune-related endocrinopathy where withhold of pembrolizumab is 
required, pembrolizumab may be resumed when AE resolves to ≤ Grade 2 and is controlled with hormonal 
replacement therapy or achieved metabolic control (in case of T1DM).   
Dosing interruptions are permitted in the case of medical/surgical events or logistical 
reasons not related to study therapy (e.g., elective surgery, unrelated medical events, 
subject vacation, and/or holidays). Subjects should be placed back on study therapy within 
4 weeks of the scheduled interruption, unless otherwise discussed with the Sponsor. The 
reason for interruption should be documented in the subject’s study record. 
 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
47 
5.3.5 Combined Denosumab and a PD-1 Agent Dosing Guidelines 
Given that denosumab’s mechanism of action presumably increases thymic output of 
autoreactive clones, it is quite possible that concurrent administration of denosumab and 
anti-PD-1 agent may increase frequency of immune-mediated adverse events caused by 
anti-PD-1 therapy (e.g. thyroiditis, rash, nephritis, pneumonitis) or, unexpectedly anti-PD-
1 agent may somehow increase frequency of AE attributed to denosumab (e.g. infections, 
ONJ, rash). It is also a possibility that the denosumab-anti-PD-1 treatment regimen may 
result in SAE that have never been previously described with any of the two drugs alone 
(e.g. stroke, myocardial infarction, anaphylactic reactions during administration of 
nivolumab or pembrolizumab). As we will describe in the Statistical Considerations 
Section (Section 8.1), we will apply continuous toxicity monitoring throughout the study 
focusing on the frequency of AE attributed to anti-PD-1 agent alone and denosumab alone, 
as well as previously undescribed AE that are definitely attributed to study drug. 
Depending on the type, frequency, and severity of AE, we may end up with decision 
regarding permanently discontinuing denosumab for either a particular subject or across 
the entire trial (and therefore the entire study) if the denosumab-anti-PD-1 agent 
combination is considered too toxic.  
5.4 
Concomitant Medications/Treatments 
All treatments that the investigator considers necessary for a subject’s welfare may be 
administered at his/her discretion, in keeping with the community standards of medical 
care.  All concomitant medications will be recorded on the case report form (CRF) 
including all prescriptions, over-the-counter (OTC), herbal supplements, and IV 
medications and fluids. If changes occur during the trial period, documentation of drug 
dosage, frequency, route, and date may also be included on the CRF. 
 
All concomitant medications received within 28 days before the first dose of trial treatment 
and 30 days after the last dose of trial treatment should be recorded.  Concomitant 
medications administered 30 days after the last dose of trial treatment should be recorded 
for SAEs and Events of Clinical Interest (ECIs) as defined in Section 7.3.3.1. 
Note: Pre-existing hypocalcemia must be corrected prior to initiating therapy with 
denosumab. Additionally, serum calcium, magnesium and phosphorus levels should be 
monitored during denosumab therapy, especially during the first 8 weeks of therapy. All 
subjects must be adequately supplemented with calcium (≥ 500mg qd) and vitamin D (≥ 
400 IU qd). 
5.4.1 Prohibited Concomitant Medications 
Subjects with pre-existing hypersensitivity to denosumab, nivolumab or pembrolizumab 
disqualifies a subject from participation in the trial. 
 
Subjects are prohibited from receiving the following therapies during the Screening and 
Treatment Phase (including retreatment for post-complete response relapse) of this trial: 
• Antineoplastic systemic chemotherapy or biological therapy  
• Immunotherapy not specified in this protocol 
• Chemotherapy not specified in this protocol 


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• Investigational agents other than nivolumab or pembrolizumab 
• Radiation therapy  
o Note:  Radiation therapy to a symptomatic solitary lesion or to the brain may 
be allowed at the investigator’s discretion.   
• Live vaccines within 30 days prior to the first dose of trial treatment and while 
participating in the trial.  Examples of live vaccines include, but are not limited to, 
the following: measles, mumps, rubella, varicella/zoster, yellow fever, rabies, 
BCG, and typhoid vaccine.  
• Systemic glucocorticoids for any purpose other than replacement/physiologic doses 
for autoimmune-related adrenal insufficiency, the management of immune-related 
events, adverse events, and as supportive care as outlined in the protocol. Study 
treatment must be held during the period of steroid administration. Subjects must 
be off steroids, if the modification allows, to resume study treatment. 
 
Subjects who, in the assessment by the investigator, require the use of any of the 
aforementioned treatments for clinical management should be removed from the trial.  
Subjects may receive other medications that the investigator deems to be medically 
necessary. 
 
The Exclusion Criteria describe other medications that are prohibited in this trial. 
Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed 
during the ongoing trial.  If there is a clinical indication for one of these or other 
medications or vaccinations specifically prohibited during the trial, discontinuation from 
trial therapy or vaccination may be required.  The investigator should discuss any questions 
regarding this with the Amgen Clinical team. The final decision on any supportive therapy 
or vaccination rests with the investigator and/or the subject's primary physician. 
 
There are no prohibited therapies during the Post-Treatment Follow-up Phase. 
5.5 
Rescue Medications and Supportive Care 
Subjects should receive appropriate supportive care measures, as deemed necessary by the 
treating investigator.  Suggested supportive care measures for the management of AEs with 
potential immunologic etiology are outlined below. Where appropriate, these guidelines 
include the use of oral or IV treatment with corticosteroids as well as additional anti-
inflammatory agents, if symptoms do not improve with administration of corticosteroids. 
Note that several courses of steroid tapering may be necessary as symptoms may worsen 
when the steroid dose is decreased. For each disorder, attempts should be made to rule out 
other causes, such as metastatic disease, bacterial or viral infection, which might require 
additional supportive care. The treatment guidelines will be applied when the investigator 
determines the events to be related to anti-PD-1 agent.  
NOTE: if after the evaluation the event is determined unrelated, the investigator does not 
need to follow the treatment guidance (as outlined below). Refer to Section 4.3.4 for dose 
modification guidelines.  


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It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, 
or skin photography as part of evaluation of the event.  
5.5.1 Management of Infusion Reactions from Pembrolizumab  
Signs and symptoms usually develop during or shortly after drug infusion and generally 
resolve completely within 24 hours of completion of infusion. The table below outlines 
guidelines for subjects who experience an infusion-related reaction to pembrolizumab 
administration. 
NCI CTCAE Grade 
Treatment 
Premedication at 
subsequent dosing 
Grade 1 
Mild reaction; infusion 
interruption not indicated; 
intervention not indicated 
Increase monitoring of vital signs as 
medically indicated until the subject is 
deemed medically stable in the opinion of the 
investigator. 
None 
Grade 2 
Requires infusion 
interruption but responds 
promptly to symptomatic 
treatment (e.g., 
antihistamines, NSAIDS, 
narcotics, IV fluids); 
prophylactic medications 
indicated for ≤ 24 hrs 
Stop Infusion and monitor symptoms. 
Additional appropriate medical therapy may 
include but is not limited to IV fluids, 
antihistamines, NSAIDS, acetaminophen, 
narcotics. 
Increase monitoring of vital signs as 
medically indicated until the subject is 
deemed medically stable in the opinion of the 
investigator. 
If symptoms resolve within one hour of 
stopping drug infusion, the infusion may be 
restarted at 50% of the original infusion rate 
(e.g., from 100 mL/hr to 50 mL/hr).  
Otherwise dosing will be held until 
symptoms resolve and the subject should be 
pre-medicated for the next scheduled dose. 
Subjects who develop Grade 2 toxicity 
despite adequate premedication should be 
permanently discontinued from further 
trial treatment administration. 
Subject may be pre-
medicated 1.5h (± 30 
minutes) prior to infusion of 
pembrolizumab with: 
 
-Diphenhydramine 50 mg po 
(or equivalent dose of 
antihistamine). 
 
-Acetaminophen 500-1000 
mg po (or equivalent dose of 
antipyretic). 
Grades 3 or 4 
Grade 3: Prolonged (i.e., 
not rapidly responsive to 
symptomatic medication 
and/or brief interruption of 
infusion); recurrence of 
symptoms following 
initial improvement; 
hospitalization indicated 
for other clinical sequelae 
(e.g., renal impairment, 
pulmonary infiltrates) 
Grade 4: Life-threatening; 
pressor or ventilatory 
support indicated 
Stop Infusion. 
Additional appropriate medical therapy may 
include but is not limited to IV fluids, 
antihistamines, NSAIDS, acetaminophen, 
narcotics, oxygen, pressors, corticosteroids, 
epinephrine. 
Increase monitoring of vital signs as 
medically indicated until the subject is 
deemed medically stable in the opinion of the 
investigator. 
Hospitalization may be indicated. 
Subject is permanently discontinued from 
further trial treatment administration. 
No subsequent dosing 
Appropriate resuscitation equipment should be available in the room and a physician readily available 
during the period of drug administration. 


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5.5.2 Contraception 
For this trial, male subjects will be considered to be of non-reproductive potential if they 
have azoospermia (whether due to having had a vasectomy or due to an underlying medical 
condition).   
 
Female subjects will be considered of non-reproductive potential if they are either:  
(1) postmenopausal (defined as at least 12 months with no menses without an alternative 
medical cause; in women < 45 years of age a high follicle stimulating hormone (FSH) 
level in the postmenopausal range may be used to confirm a post-menopausal state in 
women not using hormonal contraception or hormonal replacement therapy. In the 
absence of 12 months of amenorrhea, a single FSH measurement is insufficient);  
OR 
(2) have had a hysterectomy and/or bilateral oophorectomy, bilateral salpingectomy or 
bilateral tubal ligation/occlusion, at least 6 weeks prior to screening;  
OR 
(3) has a congenital or acquired condition that prevents childbearing. 
 
Female and male subjects of reproductive potential must agree to avoid becoming pregnant 
or impregnating a partner, respectively, while receiving study drug and for 150 days after 
the last dose of study drug by complying with one of the following:   
(1) practice abstinence† from heterosexual activity;  
OR 
(2) use (or have their partner use) acceptable contraception during heterosexual activity.   
 
Acceptable methods of contraception are‡: 
  
Single method (one of the following is acceptable): 
• 
intrauterine device (IUD) 
• 
vasectomy of a female subject’s male partner  
• 
contraceptive rod implanted into the skin 
  
Combination method (requires use of two of the following): 
diaphragm with spermicide (cannot be used in conjunction with cervical cap/spermicide) 
• 
cervical cap with spermicide (nulliparous women only)   
• 
contraceptive sponge (nulliparous women only)  
• 
male condom or female condom (cannot be used together) 
• 
hormonal contraceptive: oral contraceptive pill (estrogen/progestin pill or progestin-
only pill), contraceptive skin patch, vaginal contraceptive ring, or subcutaneous 
contraceptive injection 
  
† Abstinence (relative to heterosexual activity) can be used as the sole method of contraception if it is consistently employed as the 
subject’s preferred and usual lifestyle and if considered acceptable by local regulatory agencies and ERCs/IRBs.  Periodic abstinence 
(e.g., calendar, ovulation, sympto-thermal, post-ovulation methods, etc.) and withdrawal are not acceptable methods of contraception.  
  


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‡ If a contraceptive method listed above is restricted by local regulations/guidelines, then it does not qualify as an acceptable method of 
contraception for subjects participating at sites in this country/region. 
 
Subjects should be informed that taking the study medication may involve unknown risks 
to the fetus (unborn baby) if pregnancy were to occur during the study.  To participate in 
the study, subjects of childbearing potential must adhere to the contraception requirement 
(described above) from the day of study medication initiation (or 14 days prior to the 
initiation of study medication for oral contraception) throughout the study period up to 150 
days after the last dose of trial therapy.  If there is any question that a subject of childbearing 
potential will not reliably comply with the requirements for contraception, that subject 
should not be entered into the study. 
5.5.3 Use in Pregnancy 
If a subject inadvertently becomes pregnant while on treatment with denosumab-anti-PD-
1 agent, the subject will immediately be removed from the study.  The site will contact the 
subject at least monthly and document the subject’s status until the pregnancy has been 
completed or terminated.  The outcome of the pregnancy will be reported to Amgen without 
delay and within 24 hours to the Sponsor and within 2 working days to Amgen if the 
outcome is a SAE experience (e.g., death, abortion, congenital anomaly, or other disabling 
or life-threatening complication to the mother or newborn).  
  
The study investigator will make every effort to obtain permission to follow the outcome 
of the pregnancy and report the condition of the fetus or newborn to the Sponsor. If a male 
subject impregnates his female partner, the study personnel at the site must be informed 
immediately and the pregnancy reported to the Sponsor and to Amgen, as described above 
and in Section 7.3.3. 
5.5.4 Overdose of Pembrolizumab 
There is no information on overdosage of pembrolizumab. 
5.5.5 Overdose of Nivolumab 
There is no information on overdosage of nivolumab. 
5.5.6 Overdose of Denosumab 
There is no experience with overdosage of denosumab. 
5.6 
Other Modalities or Procedures 
A mandatory tumor biopsy will be performed on day 22 of the study, after 3 injection of 
denosumab and immediately prior to the administration of the first dose of PD-1 inhibitor. 
Depending on the location of the tumor, the biopsy can be excisional or punch and 
performed as an outpatient procedure at the bedside (if palpable; e.g. subcutaneous, 
cutaneous, or palpable lymph node or CT-guided by interventional radiology). In addition, 
an optional biopsy can be offered to subjects on week 16 (i.e., should be performed within 
1 week of scheduled visit at week 16). 
 
Up to 100 mL of peripheral blood will be collected for the purpose of analyzing the 
translational endpoints outlined in section 1.10.1.2. The reasons for the high amount of 


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UNIVERSITY OF NORTH CAROLINA 
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blood to be collected are many-fold: (a) the immune cell subsets to be analyzed and 
enumerated are rare events, especially in adults (thymic input and output, MDSC), (b) 
peripheral blood lymphopenia is frequent in subjects with metastatic melanoma and an 
adverse prognostic factor 68, (c) assays will be performed in batched samples on the same 
day to avoid artifacts associated with assay variability; this implies that for each time point 
PBMCs will be isolated and cryopreserved, a procedure which is inherently associated with 
at least 30% loss of PMBCs upon thawing and reconstitution. Therefore, to compensate for 
these challenges, and on condition that subjects meet certain inclusion criteria [hemoglobin 
is ≥10g/dL, absolute lymphocyte count (ALC) is ≥1,000 cells/mm3, absent coronary artery 
disease], a higher amount of peripheral blood should be collected to be able to measure the 
primary translational endpoints of the study. 
 
A frequent scenario that we may encounter with respect to isolating adequate numbers of 
PBMCs is as follows. Subjects may have ALC approximately 1,000 cells/mm3. Under the 
optimal conditions of Ficoll separation, processing of 100 mL of peripheral blood will yield 
approximately 100 million PMBCs. Of these cells, approximately 50-70 million will be 
suitable for downstream analysis following thawing and reconstitution on the day of the 
assay(s). The following are the minimum-optimal requirements (in millions of cells) for 
each assay: 
(a) items 1, 2, 5, 6, 7, and 8 of section 1.10.1.2 15 million cells; no further 
processing is required. 
(b) item 3 of section 1.10.2.1 (CD4+ and CD8+ sjTREC). The assay requires 
approximately 3 µg of genomic DNA in duplicates (at least). An adequate DNA 
extraction of genomic DNA from PBMCs yields approximately 4-6 µg of DNA per 
one million cells. We will perform isolation of CD4+ and CD8+ positive cells; they 
are roughly 5-30% of CD8+ T cells and 25-60% of CD4+ T cells in PBMCs. In 
addition, up to 20% of reconstituted PBMCs following thawing will not be reliably 
sorted into CD4+ and CD8+ compartments by FACS analysis. This means that for 
every 20 million reconstituted cells we will be able to isolate approximately 0.8 
million CD8+ cells (worst case scenario) and 4 million CD4+ cells (worst case 
scenario). Thus, approximately 3.2 µg of DNA can be isolated for CD8+ sjTREC 
analysis (worst case scenario) and 16 µg of DNA can be isolated from CD4+ 
sjTREC. Therefore, 38 million PBMCs are required to ensure enough CD8+ sorted 
cells to perform the sjTREC analysis in duplicates. 
(c) item 4 (CD4+ and CD8+ selected cells for immune cell receptor repertoire 
analysis). The assay requires 100 ng of RNA. Given that abundance of RNA in µg 
is approximately 5 times lower than that of DNA, one million PBMCs yields 
approximately 0.75-1 µg of RNA. CD4+ and CD8+ cells will also be sorted 
following thawing and reconstitution. Thus, 5 million cells will yield 200 ng of 
RNA from CD4+ sorted PBMCs and 1,000 ng of RNA from CD8+ sorted PBMCs. 
Given that DNA/RNA extraction will occur sequentially on the same batch of 
PBMCs sorted for CD4+ and CD8+ cells, we will isolate more than enough RNA 
using the 38 million PBMC batch for DNA extraction. 
 


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In summary, for a subject whose absolute lymphocyte count is approximately 1,000 
cells/mm3, approximately 53 million cells are necessary to perform the proposed 
downstream analyses.  
5.7 
Duration of Therapy 
In the absence of treatment delays due to AEs, treatment with study medications may 
continue for up to 1 year or until: 
• Disease progression, 
• Intercurrent illness that prevents further administration of treatment, 
• Unacceptable adverse event(s), 
• Pregnancy, 
• Subject decides to withdraw from study treatment, or 
• General or specific changes in the subject’s condition render the subject 
unacceptable for further treatment in the judgment of the investigator. 
 
Subjects receiving clinical benefit at the conclusion of the study assessment period (after 1 
year) may continue anti-PD-1 agent as per standard of care.  
5.8 
Duration of Follow Up 
Subjects who have not progressed on PD-1 inhibitor plus denosumab after a year of 
treatment will be followed every 3 months for up to 2 years (i.e., a total of 3 years since 
study initiation) with phone calls or visits and then annually until death via medical record 
abstraction or phone calls. The following information will be recorded: (a) details regarding 
systemic treatment for their melanoma that they are currently on (type, response if any, 
duration on treatment), (b) whether they have progressed (and when) from current PD-1 
inhibitor treatment, (c) whether treatment-related AEs that subjects may have developed 
during the year of denosumab- PD-1 inhibitor treatment have resolved, and when, (d) 
whether they are alive or not; if dead, precise date of death.  
 
Subjects who have progressed on PD-1 inhibitor plus denosumab within the year of the 
study duration will be followed every 3 months for up to 3 years since study initiation with 
phone calls or standard of care visits and then annually until death via medical record 
abstraction or phone calls. The following information will be recorded: (a) systemic 
treatment for their melanoma they are currently on (type, response if any, duration on 
treatment, treatment-related AEs), (b) whether treatment-related AEs that subjects may 
have developed during the year of denosumab plus PD-1 inhibitor treatment have resolved, 
and when, (c) whether they are alive or not; if dead, precise date of death.  
 
Subjects removed from study treatment for unacceptable AEs will be followed for 
resolution or stabilization of the adverse event(s). All subjects (including those withdrawn 
for AEs) should be followed after removal from study treatment as stipulated in the 
protocol.   
 
Survival follow up will continue annually until death via medical record abstraction or 
phone calls.  


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5.9 
Removal of Subjects from Protocol Therapy 
Subjects will be removed from protocol therapy and the PI notified when any of the criteria 
listed in section 4.7 apply. The reason for discontinuation of protocol therapy will be 
documented on the electronic CRF (eCRF).   
 
In case a subject decides to prematurely discontinue protocol therapy (“refuses 
treatment”), the subject should be asked if she or he may still be contacted for further 
scheduled study assessments. The outcome of that discussion should be documented in 
both the medical records and in the eCRF. 
 
High number of subject withdrawals from study can render the study un-interpretable; 
therefore, unnecessary withdrawal of subjects will be avoided. 
5.10 
Study Withdrawal 
If a subject decides to withdraw from the study (and not just from protocol therapy) an 
effort should be made to complete and report study assessments as thoroughly as possible.  
At the time of withdrawal, the investigator should attempt to establish the reason for the 
study withdrawal as completely as possible. The option of contacting the next of kin should 
be available. 
• 
The subject should be asked if they are willing to allow for the abstraction of 
relevant information from their medical record in order to meet the long term follow 
up (e.g., survival) objectives outlined in the protocol.   
• 
A complete final evaluation at the time of the subject’s study withdrawal should be 
obtained with an explanation of why the subject is withdrawing from the study.   
• 
If the subject is noncompliant and does not return for an end-of-study follow-up 
assessment, this should be documented in the eCRF.  
• 
If the reason for removal of a subject from the study is an AE, the principal specific 
event will be recorded on the eCRF.  
 
Excessive subject withdrawals from protocol therapy or from the study can render the study 
un-interpretable; therefore, unnecessary withdrawal of subjects should be avoided. 
 
 


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UNIVERSITY OF NORTH CAROLINA 
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55 
6.0 
DRUG INFORMATION 
6.1 
Denosumab 
6.1.1 Indications 
• 
Prevention of skeletal-related events in subjects with bone metastases from solid 
tumors. 
• 
Treatment of adults and skeletally mature adolescents with giant cell tumor of bone 
that is unresectable, or where surgical resection is likely to result in severe 
morbidity. 
• 
Treatment of hypercalcemia of malignancy refractory to bisphosphonate therapy. 
6.1.2 Mechanism of action 
Denosumab binds to RANKL, a transmembrane or soluble protein essential for the 
formation, function and survival of osteoclasts, the cells responsible for bone resorption, 
thereby modulating calcium release from bone. Increased osteoclast activity, stimulated by 
RANKL, is a mediator of bone pathology in solid tumors with osseous metastases. 
Denosumab prevents RANKL from activating its receptor, RANK, on the surface of 
osteoclasts, their precursors, and osteoclast-like giant cells. 
6.1.3 Product description 
Denosumab is a human IgG2 monoclonal antibody that binds to human RANKL. 
Denosumab has an approximate molecular weight of 147 kDa and is produced in 
genetically engineered mammalian (Chinese hamster ovary) cells. 
6.1.4 Supplier/How Supplied 
Denosumab will be supplied as a sterile, clear, colorless to slightly yellow, 
preservative-free liquid, in single-use 3.0 mL glass vials containing a deliverable dose of 
1.7 mL. 
6.1.5 Storage and Handling 
Store denosumab in a refrigerator at 2°C to 8°C (36°F to 46°F) in the original carton. Do not 
freeze. Once removed from the refrigerator, denosumab must not be exposed to 
temperatures above 25°C/77°F or direct light and must be used within 24 hours. Protect 
from direct light and heat. Avoid vigorous shaking of denosumab. 
6.1.6 Dose and route of administration 
Denosumab is intended for s.c. route only and should not be administered IV, 
intramuscularly, or intradermally. 
 
The recommended dose is 120 mg administered as a s.c. injection every 4 weeks in the 
upper arm, upper thigh, or abdomen. In this study we will administer additional loading 
doses of 120 mg denosumab on day 8 and day 22 (after Amendment 1). 
 


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56 
6.1.7 Possible side effects 
• 
Hypersensitivity. Clinically significant hypersensitivity has been reported with 
denosumab. If an anaphylactic or other clinically significant allergic reaction occurs, 
initiate appropriate therapy and discontinue denosumab therapy permanently. 
 
• 
Hypocalcemia. Denosumab can cause severe symptomatic hypocalcemia, and fatal 
cases have been reported. Monitor calcium levels through denosumab therapy, 
especially in the first 8 weeks of initiating therapy. We will advise all subjects to take 
daily supplements containing 1,000 mg calcium, or more, and 400 IU of vitamin D, or 
more. 
 
• 
Osteonecrosis of the Jaw (ONJ). ONJ manifesting as jaw pain, osteomyelitis, bone 
erosion, tooth or periodontal infection, tooth ache, gingival ulceration, or gingival 
erosion. Consider temporary discontinuation of denosumab therapy if an invasive 
dental procedure must be performed.  
 
• 
Atypical subtrochanteric and diaphyseal femoral fracture. These fractures can occur 
anywhere in the femoral shaft. A number of reports note that subjects were also 
receiving treatment with glucocorticoids at the time of fracture. Subjects should be 
advised to report new or unusual thigh, hip, or groin pain. 
 
• 
Hypercalcemia following treatment discontinuation in subjects with growing 
skeletons. 
 
• 
Embryo-fetal toxicity. Denosumab can cause fetal harm when administered to a 
pregnant woman. Advise females of reproductive potential to use highly effective 
contraception during therapy and for at least 150 days thereafter. 
6.1.8 Handling and Disposal 
Please see policy on hazardous drugs:  
http://intranet.unchealthcare.org/intranet/hospitaldepartments/safetynet/policies/hazardou
sdrugs.pdf 
 
Local requirements for disposal of hazardous drugs should be followed at each 
participating clinical site. 
6.1.9 Precautions 
Pre-existing hypocalcemia must be corrected prior to initiating therapy with denosumab. 
See section above possible side effects Hypocalcemia for details. 
 
For additional information, please refer to the full prescribing information on denosumab 
is available at: http://pi.amgen.com/united_states/xgeva/xgeva_pi.pdf 
 


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57 
6.2 
Pembrolizumab 
Pembrolizumab will be provided by subject’s health insurance. Clinical supplies will be 
affixed with a clinical label in accordance with regulatory requirements. 
6.2.1 Description 
Pembrolizumab solution for infusion is a sterile, non-pyrogenic aqueous solution supplied 
in single-use type I glass vial containing 100 mg/4 mL of pembrolizumab (manufactured 
using the fully formulated drug substance with L-histidine as a buffering agent, polysorbate 
80 as a surfactant, and sucrose as a stabilizer/tonicity modifier). The product is 
preservative-free solution which is essentially free of extraneous particulates.  
6.2.2 Supplier/How Supplied 
Pembrolizumab is supplied as single-use 100 mg vials containing a sterile, non-pyrogenic, 
clear to opalescent aqueous solution (25 mg/mL), or as single-use 50 mg vials containing 
lyophilized powder in a single-dose vial for reconstitution). Pembrolizumab solution for 
infusion is formulated in 10mM histidine buffer, pH 5.2-5.8, containing 7% sucrose and 
0.02% polysorbate 80, supplied in Type I glass vials with a cap color of red, salmon, or 
blue.  
6.2.3 Storage and Handling Requirements and Dispensing 
Clinical supplies must be stored in a secure, limited-access location under refrigerated 
conditions (2°C to 8°C). 
Receipt and dispensing of trial medication must be recorded by an authorized person at the 
trial site. 
Clinical supplies may not be used for any purpose other than that stated in the protocol. 
6.2.4 Preparation of Infusion Solution 
Aseptic technique must be strictly observed throughout the preparation procedure, 
preferably in a biologic safety cabinet or hood since no anti-microbial preservative is 
present in the solutions.  
Equilibrate required number of pembrolizumab vials to room temperature. 
The preferred method of dose preparation is the volumetric method, gravimetric method is 
not permitted.  
Choose a suitable infusion bag size so that the following conditions are met: 
o Concentration of pembrolizumab is between 1 mg/mL and 10 mg/mL 
o The infusion volume to bag capacity ratio should not be less than 0.3.  In other 
words, the bag must be filled to at least 30% of its capacity.   
Choose a suitable infusion bag material. The bag may be empty, or it may contain normal 
saline. The following infusion bag materials are compatible with pembrolizumab:  
 PVC plasticized with DEHP 
 Non-PVC (polyolefin) 
 EVA 
 PE lined polyolefin 
      *Contact Sponsor for materials not listed above 
 


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UNIVERSITY OF NORTH CAROLINA 
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58 
Calculate the volume of pembrolizumab and normal saline required to prepare the infusion 
(admixture) bag 
 
Volume of pembrolizumab (mL) = required dose amount (mg) / 25 (mg/mL) 
 
Volume of normal saline = total infusion volume – volume of pembrolizumab from above 
 
If a bag pre-filled with normal saline is being used, remove the excess volume of normal 
saline using a sterile syringe (Polypropylene, latex-free) attached to a suitable needle. Keep 
in consideration the excess bag fill volume as well as the volume of reconstituted 
pembrolizumab to be added to the bag to prepare the infusion solution. 
If an empty bag is being used, withdraw the necessary volume of normal saline from 
another appropriate bag and inject into the empty bag. Keep in consideration the volume 
of reconstituted pembrolizumab to be added to the bag to prepare the infusion solution. 
 
Withdraw the required volume of pembrolizumab from the vial(s) (up to 4 mL from each 
vial) using a sterile syringe attached to a suitable needle. The vial(s) may need to be 
inverted to remove solution. 
 
Volume of pembrolizumab (mL) = required dose amount (mg) / 25 (mg/mL) 
 
Note: If it is necessary to use several vials, it is advisable to withdraw from several vials 
into a suitable size single use syringe using a new needle for each vial.  
 
Add the required pembrolizumab into the infusion IV bag containing normal saline and 
gently invert the bag 10-15 times to mix the solution. 
 
Pembrolizumab solutions may be stored at room temperature for a cumulative time of up 
to 6 hours. This includes room temperature storage of admixture solutions in the IV bags 
and the duration of infusion.  
 
In addition, IV bags may be stored under refrigeration at 2 °C to 8 °C (36 °F to 46 °F) for 
up to 20 hours. If refrigerated, allow the IV bags to come to room temperature prior to use.   
Do not freeze the pembrolizumab infusion solution. 
Discard any unused portion left in the vial as the product contains no preservative. 
6.2.5 Method of Administration 
Pembrolizumab infusions should be administered in 30 minutes, with a window of -5 and 
+10 minutes, using an infusion pump. A central catheter is not required for infusion; 
however, if a subject has a central venous catheter in place, it is recommended that it be 
used for the infusion. The following infusion set materials are compatible with 
pembrolizumab: 
 
o PVC and tri-(2-ethylhexyl) trimellitate (TOTM) infusion set 
o Polyethylene lined PVC infusion set 
o PVC Infusion set that is plasticized using Di-2-ethylhexyl Terephthalate (DEHT) 


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UNIVERSITY OF NORTH CAROLINA 
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 July 7, 2022 
59 
o Polyurethane set 
 
A sterile, non-pyrogenic, low-protein binding 0.2 to 5 µm in-line filter made of 
polyethersulfone (PES) must be used during administration to remove any adventitious 
particles.  If the infusion set does not contain 0.2 to 5 µm in-line filter, it is recommended 
to use 0.2 to 5 µm add-on filter which may contain an extension line (Note: the materials 
of the extension line and filter should be as mentioned above). 
Attach the infusion line to the pump and prime the line, either with normal saline (at least 
25 mL) or with infusion solution as per local SOP, before starting the infusion. 
Infuse pembrolizumab over approximately 30 minutes, with a window of -5 and +10 
minutes, through a peripheral line or indwelling catheter.  
Ensure the entire contents of the bag are dosed and all remaining drug solution in the line 
is administered according to institutional guidelines for saline flushing.  
Document volume administered according to data entry guidelines. 
 
In case of infusion reactions, infusion rate may differ; refer to protocol for specific 
instructions.  
Whenever possible, the lowest infusion rate should be used that will allow completion 
of the infusion within the 30 minutes. Maximum rate of infusion should not exceed 6.7 
mL/min. through a peripheral line or indwelling catheter. However, when it is 
necessary to infuse a larger volume (i.e. 250 mL), the flow rate may go as high as 10 
mL/min (maximum) in order to keep the infusion within the window as defined above.  
 
Do not co-administer other drugs through the same infusion line. 
 
Unused infusion solution for injection should not be used for another infusion of the same 
subject or different subject. 
6.2.6 Storage and Stability 
Pembrolizumab (MK-3475) Solution for Infusion, 100 mg/ 4 mL vial: pembrolizumab 
solution for Infusion vials should be stored at refrigerated conditions (2 – 8 °C) and 
protected from light. 
 
Note: vials should be stored in the original box to ensure the drug product is protected from 
light. 
 
Pembrolizumab infusion solutions should be prepared in 0.9% Sodium Chloride Injection, 
USP (normal saline) or regional equivalent or 5% Dextrose Injection, USP (5% dextrose) 
or regional equivalent and the final concentration of pembrolizumab in the infusion 
solutions should be between 1 mg/mL and 10 mg/mL. 
Please note, the preferred diluent is 0.9% sodium chloride; 5% dextrose is only permissible 
if normal saline is not available. 
Pembrolizumab should not be mixed with other diluents.  
Pembrolizumab solutions may be stored at room temperature for a cumulative time of up 
to 4 hours. This includes room temperature storage of admixture solutions in the IV bags 
and the duration of infusion 


LCCC 1620 
 
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60 
In addition, IV bags may be stored under refrigeration at 2°C to 8°C (36°F to 46°F) for up 
to a cumulative time of 20 hours. If refrigerated, allow the IV bags to come to room 
temperature prior to use. 
Parenteral drug products should be inspected visually for particulate matter and 
discoloration prior to administration. Discard the drug product vial if extraneous particulate 
matter other than translucent to white proteinaceous particles is observed. 
Sites should follow their SOPs for drug transport and delivery, with all possible effort to 
minimize agitation of the drug product between the pharmacy and the clinic. 
Do not use pembrolizumab if discoloration is observed. 
Do not shake or freeze the vial(s). 
Do not administer the product as an intravenous (IV) push or bolus. 
Do not combine, dilute or administer it as an infusion with other medicinal products. 
Any deviation from this guidance must be discussed with sponsor 
6.2.7 Return and Retention 
The investigator is responsible for keeping accurate records of the clinical supplies 
received from Merck or designee, the amount dispensed to and returned by the subjects 
and the amount remaining at the conclusion of the trial.  
 
Upon completion or termination of the study, all unused and/or partially used 
investigational product will be destroyed at the site per UNC IDS drug destruction policy. 
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. 
6.3 
Return and Retention of Study Medications 
The investigator is responsible for keeping accurate records of the clinical supplies 
received from Amgen and or designee, the amount dispensed to and returned by the 
subjects and the amount remaining at the conclusion of the trial.  
 
Upon completion or termination of the study, all unused and/or partially used denosumab 
will be destroyed at the site per UNC IDS drug destruction policy. It is the Principal 
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. 
 
6.4 
Nivolumab 
Nivolumab will be administered per standard of care following the institutional guidelines. 
Please consult the prescribing information for nivolumab available at: 
https://packageinserts.bms.com/pi/pi_opdivo.pdf 
 
 


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6.4.1 Description 
Nivolumab injection drug product is a sterile, non-pyrogenic, single-use, isotonic aqueous 
solution formulated at 10 mg/10 mL. The vials supplied contain 100 mg.  Nivolumab is a 
sterile, preservative-free, non-pyrogenic clear to opalescent, colorless to pale-yellow liquid 
that may contain light (few) particles for IV administration. 
6.4.2 Supplier/How Supplied 
Nivolumab is supplied as single-use 240 mg/24 mL (10 mg/mL) vials containing a sterile, 
non-pyrogenic, clear to opalescent, colorless to pale-yellow solution in a single-dose vial. 
6.4.3 Packaging and Labeling 
Primary Packaging (Volume)/Label type: Carton of 5 or 10 vials. 
Secondary Packaging (Qty)/Label type: 10-mL Type 1 flint glass vials stoppered with butyl 
stoppers and sealed with aluminum seals. 
6.4.4 Storage and Handling 
Store nivolumab under refrigeration at 2°C to 8°C (36°F-46°F) in the original package until 
time of use. Protect from light and freezing. Do not shake the vial. 
 
If stored in a glass front refrigerator, vials should be stored in the carton. Recommended 
safety measures for preparation and handling of nivolumab include laboratory coats and 
gloves. 
 
Nivolumab solution for injection can be stored after preparation at room temperature for 
no more than 4 hours. This includes room temperature storage of the infusion in the IV 
container and time for administration of the infusion OR under refrigeration at 2°C to 8°C 
(36°F-46°F) for nor more than 24 hours from the time of infusion preparation. 
6.4.5 Dose, Schedule and Administration 
Nivolumab will be given every four weeks at a dose of 480 mg to be administered as an IV 
infusion per institutional guidelines. 
 
There are no premedications recommended for nivolumab on the first cycle. 
 
Subjects should be carefully monitored for infusion reactions during nivolumab 
administration. If an acute infusion reaction is noted, subjects should be managed 
according to institutional guidelines. 
 
Doses of nivolumab may be interrupted, delayed, or discontinued depending on how well 
the subject tolerates the treatment. 
6.4.6 Preparation 
Follow institutional guidelines for nivolumab preparation or see the nivolumab prescribing 
information for preparation instructions. Nivolumab is to be administered as an IV infusion 
through a 0.2-micron to 1.2-micron pore size, low-protein binding polyether sulfone (PES) 
membrane in-line filter at the protocol-specified dose. It is not to be administered as an IV 


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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 1 mg/mL. Care must be taken to assure sterility of the 
prepared solution as the product does not contain any antimicrobial preservative or 
bacteriostatic agent. 
6.4.7 Handling and Disposal  
Please see UNC policy on hazardous drugs:  
http://intranet.unchealthcare.org/intranet/hospitaldepartments/safetynet/policies/hazardou
sdrugs.pdf 
6.4.8 Adverse Events Associated with Nivolumab 
The adverse events listed below have been reported in subjects receiving nivolumab.  
• Immune-mediated pneumonitis: Defined as requiring use of corticosteroids and no 
clear alternate etiology, including fatal cases have occurred with nivolumab 
treatment. 
• Immune-mediated colitis: Defined as requiring use of corticosteroids and no clear 
alternate etiology, including fatal cases have occurred with nivolumab treatment. 
• Immune-mediated hepatitis: Defined as requiring use of corticosteroids and no clear 
alternate etiology have occurred with nivolumab treatment. 
• Immune-mediated endocrinopathies: Hypophysitis, adrenal insufficiency, hypo- 
and hyper-thyroidism, and type I diabetes mellitus have occurred with nivolumab 
treatment. 
• Immune-mediated nephritis and renal dysfunction: Defined as renal dysfunction or 
grade ≥2 increased creatinine, requirement for corticosteroids, and no clear 
alternate etiology, can occur with nivolumab treatment. 
• Immune-mediated rash: Severe rash (including rare cases of fatal toxic epidermal 
necrolysis) can occur with nivolumab treatment. 
• Immune-mediated encephalitis: Withhold nivolumab in subjects with new-onset 
moderate to severe neurologic signs or symptoms. 
• Complications of allogeneic human stem cell transplant after nivolumab: Monitor 
for hyper acute graft-versus-host disease (GVHD). Transplant related mortality has 
occurred. 
• Infusion reactions: Severe infusion reactions have been reported in <1.0% of 
subjects in clinical trials of nivolumab.  
 
 
 
 
 
 
 
 


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7.0 
EVALUATIONS AND ASSESSMENTS 
7.1 
Time and Events Table   
 
Assessments1 
Pre-
Study1 
Day-
21 to -
0 
Study Treatment (cycle 1 = 21 days, cycle 2+n = 28 days) 
End 
Long-term 
follow up21 
C1D1 
C1D8 
C2D1 
C2D2 
C3D
1 
C4D1 
C5D1 
C6D1 –  
C13D1 
of Tx 
Wk1 
D1±1
d 
Wk2 
D8±1
d 
Wk4 
D22±2
d 
Wk4 
D23-2 
days 
through 
+1 day 
Wk8 
D50±
3d 
Wk12 
D78±7
d 
Wk16 
D106±7d 
Wks17-4920 
(Wks22/28/3
4/ 
40/46±7 
days) 
Wk52 
 
Informed consent 
× 
 
 
 
 
 
 
 
 
 
 
History, PE2 
× 
× 
 
× 
 
× 
× 
× 
× 
× 
× 
Serum pregnancy test 
×3 
 
 
 
 
 
 
 
 
 
 
ECOG Performance status 
× 
×4 
 
× 
 
× 
× 
× 
× 
× 
 
Concomitant medications review 
× 
× 
× 
× 
 
× 
× 
× 
× 
× 
 
CBC with differential5  
× 
×4 
 
× 
 
 
× 
 
× 
× 
 
Serum chemistries6 
× 
×4,6 
×6 
× 
 
× 
× 
× 
× 
× 
 
Endocrine tests7 
× 
× 
 
× 
 
× 
× 
× 
× 
× 
 
Urinalysis 
× 
×4 
 
× 
 
 
× 
 
× 
× 
 
Toxicity assessment8 
× 
× 
× 
× 
 
× 
× 
× 
× 
× 
 
Brain MRI9 
× 
 
 
 
 
 
 
 
 
 
 
Whole body CT10 
× 
 
 
 
 
 
 
× 
×10 
×10 
 
Denosumab11 
 
× 
× 
× 
 
× 
× 
× 
× 
 
 
Nivolumab12 
 
 
 
 
×12 
× 
× 
× 
×12, 20 
×13 
 
Peripheral blood mononuclear cells15 
 
× 
 
 
× 
 
 
× 
× 
× 
 
Hydration prior to blood draw14 
 
× 
 
 
× 
 
 
 
× 
× 
 
Archival tissue16, 17 
×16, 17 
 
 
 
×18 
 
 
×19 
 
 
×17 
Tumor Biopsies17 
×17 
Dental examination 
×22 
 
 
 
 
 
 
 
 
 
 


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64 
Footnotes to Time and Events Table 
 
1. All screening labs to be obtained within 21 days prior to registration.  All study visits should be 
performed within ±1 day for the first two weeks of study therapy. For the C2D1 visit the window is ±2 
days, then ±3 days for assessments scheduled on Day 50 (Week 8) and then ±7 days for all subsequent 
visits. 
2. Complete history/physical at baseline only, thereafter focused history/physical exam on 
symptoms/toxicity. 
3. Serum pregnancy test should be performed within 72 hours prior to the first dose of denosumab. 
4. All screening labs to be obtained within 21 days prior to registration.  
5. Will be performed at baseline and every other nivolumab infusion starting from C2D1, as per standard 
of care. 
6. Include sodium, potassium, chloride, CO2, BUN, creatinine, glucose, calcium, albumin, total protein, 
total bilirubin, AST, ALT, alkaline phosphatase, magnesium, phosphorus, LDH, amylase and lipase. All 
listed serum chemistries will be performed before each nivolumab infusion as per standard of care.  At 
Cycle 1 Day 8, magnesium, calcium, and phosphorus only. 
7. Include cortisol levels, free T4 (fT4)/TSH, prolactin (women only), and testosterone (males only). 
8. Toxicity assessments per NCI-CTCAE version 5.0. 
9. Is performed at baseline; on future visits only if clinically indicated, as per standard of care. 
10. Includes CT of the neck (if applicable), chest, abdomen, pelvis with IV contrast. Scans are performed at 
baseline, week 16, and then every 12 weeks thereafter (i.e., weeks 28, 40, and 52). Scans may be 
performed within ±7 days of the scheduled visit. 
11. Denosumab will be administered s.c. as a flat dose, 120 mg every 4 weeks, as per FDA-approved dose, 
for up to a year until intolerable toxicity or disease progression occurs. Extra 120 mg loading doses of 
denosumab will be administered on C1D8 and C2D1. See Dosing schema in section 4.2.1. Denosumab 
will not be continued beyond the one-year duration of the trial, irrespective of treatment response. 
12. Nivolumab will be administered every 4 weeks, 480 mg, for up to a year, starting on C2D2, until 
intolerable toxicity or disease progression occurs. See Dosing schema in section 4.2.1. Note that first 
dose of nivolumab may be given on day of biopsy depending on recovery from biopsy. 
13. Following completion of the trial at 1 year and on condition that there is no disease progression, 
nivolumab can be continued at the opinion of the investigator, as per standard of care guidelines. 
14. Subjects should receive hydration with 500 mL of normal saline per discretion of treating investigator 
prior to, in between or, following blood draw for correlative studies on week 1, week 4, week 16, week 
28, week 40 and at the end of treatment visit.  
15. PBMCs will be isolated from peripheral blood (approximately 100 mL at each time point) using the 
Ficoll isolation technique; an aliquot will be used for multiparameter flow cytometry, another for 
immune receptor repertoire analysis, and another for sjTCR excision cycle analysis. Samples will be 
collected at baseline on D1/wk1 and on D22/wk4, D106/wk16, wk28, wk40 and end of treatment.  500 
mL of normal saline will be administered through IV either prior to collection, in between collection of 
study tubes, or following collection of all tubes. 
16. Archival tissue or fresh tumor will be collected at baseline and a mandatory excisional biopsy will be 
performed on D22 of the study, after the third denosumab injection and before the first PD-1 inhibitor 
infusion. An optional excisional biopsy should be obtained on D106 (wk16). Tumor tissue will be 
submitted to the Tissue Pathology Laboratory (TPL) for IHC and multicolor IF. If archived tumor blocks 


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65 
are not available, subject can still be enrolled on condition that he/she has a tumor biopsy prior to study 
enrollment. 
17. Optional tumor tissue samples from standard of care surgical procedures may be collected for research 
purposes while the subjects are on study. These include samples that may have been collected after study 
enrollment, but prior to study biopsies, from any surgical procedure that occurs during study enrollment, 
and those procedures that may occur after study treatment completion as long as the subject is still on 
study. NOTE: For subjects who are withdrawn from LCCC1620, subjects must be re-consented and 
placed back on the study for attainment of optional tumor tissue samples from standard of care surgical 
procedures. These subjects will then remain on study for long term follow up.  
18. Mandatory (excisional, punch or CT-guided), performed 24 – 48 hours after third denosumab injection 
and prior to nivolumab infusion. 
19. Optional 
20. Following radiographic disease assessment on week 16, subjects will be seen by the investigator on 
every other nivolumab infusion (i.e., every 8 weeks on D1 of weeks 24, 32, 40, 48 and at the end of 
treatment visit at week 52) for safety assessment and on every fourth nivolumab infusion (i.e., 16 weeks) 
for response assessment for up to a year. See sections identified below for details of scheduled 
assessments.  
21. Subjects who have not progressed on nivolumab plus denosumab after a year of treatment will be 
followed every 3 months for up to 2 years (i.e. a total of 3 years since study initiation) with at least phone 
calls and then annually until death via medical record abstraction or phone calls. The following 
information will be recorded: (a) details regarding systemic treatment for their melanoma that they are 
currently on (type, response if any, duration on treatment, treatment-related side effects), (b) whether 
they have progressed (and when) from current PD-1 inhibitor treatment, (c) whether treatment-related 
adverse events that subjects may have developed during the year of denosumab plus nivolumab treatment 
have resolved, and when, (d) whether they are alive or not; if dead, precise date of death. Subjects who 
have progressed on nivolumab plus denosumab within the year of the study duration will be followed 
every 3 months for up to 3 years since study initiation with at least phone calls and then annually until 
death via medical record abstraction or phone calls. The following information will be recorded: (a) 
systemic treatment for their melanoma they are currently on (type, response if any, duration on treatment, 
treatment-related side effects), (b) whether treatment-related AEs that subjects may have developed 
during the year of denosumab plus nivolumab treatment have resolved, and when, (c) whether they are 
alive or not; if dead, precise date of death. Survival follow up will continue annually until death via 
medical record abstraction or phone calls.  
22. Subjects with poor dentition or other dental issues that, in the opinion of the treating physician, may 
increase the risk of osteonecrosis of the jaw, should be examined and cleared by dentist prior to treatment 
initiation. 
 
7.2 
Follow-up Assessments 
Subjects who have not progressed on PD-1 inhibitor plus denosumab after a year 
of treatment will be followed every 3 months for up to 2 years (i.e. a total of 3 years 
since study initiation) with phone calls or standard of care visits. The following 
information will be recorded: (a) details regarding systemic treatment for their 
melanoma that they are currently on (type, response if any, duration on treatment, 
treatment-related side effects), (b) whether they have progressed (and when) from 
current PD-1 inhibitor treatment, (c) whether treatment-related AEs that subjects 
may have developed during the year of denosumab plus PD-1 inhibitor treatment 
have resolved, and when, (d) whether they are alive or not; if dead, precise date of 


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UNIVERSITY OF NORTH CAROLINA 
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66 
death. Subjects who have progressed on PD-1 inhibitor plus denosumab within the 
year of the study duration will be followed every 3 months for up to 3 years since 
study initiation with phone calls or standard of care visits. The following 
information will be recorded: (a) systemic treatment for their melanoma they are 
currently on (type, response if any, duration on treatment, treatment-related side 
effects), (b) whether treatment-related AEs that subjects may have developed 
during the year of denosumab plus PD-1 inhibitor treatment have resolved, and 
when, (c) whether they are alive or not; if dead, precise date of death. 
7.3 
Correlative Studies Procedures 
As part of this protocol, peripheral blood will be collected for quantification of 
certain hormones using ELISA and enumeration of immune cell subsets using 
multiparameter flow cytometry whereas tumor tissue will be collected for 
assessment of TILs, both effector and immunoregulatory, and treatment effects of 
the denosumab and PD-1 inhibitor combination at the tumor site. Detailed 
description of correlative studies in peripheral blood and tumor tissues is available 
in section 1.10. Rationale regarding the amount of blood requested per each time 
point is available in section 4.6. No dedicated genetic analysis will be performed 
beyond the standard of care. The sections below describe details regarding time 
points for specimen collection, amount of tissue to be collected at each time point 
and procedures for specimen collection, handling, and storage. 
7.3.1 Peripheral Blood 
Peripheral blood will be collected at baseline (day 1, week 1), day 22 (week 4), day 
105 (week 16), week 28, week 40 and end of treatment for multiparameter flow 
cytometric analysis of immune cell subsets, signal joint TCR excision circle 
analysis (sjTREC) of CD4+ and CD8+ sorted T cells, immune cell receptor (TCR 
and B-cell receptor) repertoire analysis of CD4+ and CD8+ sorted T cells, and 
ELISA studies (RANKL and OPG serum levels). 100 mL of peripheral blood will 
be collected for non-ELISA studies and 5 mL will be collected for ELISA studies.  
7.3.1.1 Peripheral blood Mononuclear Cell Isolation 
• Collect peripheral blood in ten 10mL green-top (sodium heparin) tubes (BD 
Vacutainer, Catalog no. 366480). Mix the blood by gently converting the 
tube few times right after the blood is drawn. 
• Transport green top tubes at ambient temperature on the same day of 
collection to the Tissue Procurement Facility, Lineberger Comprehensive 
Cancer Center (LCCC), for PBMC isolation and cryopreservation storage. 
Peripheral blood collection will be limited to Monday-Thursday. 
• Ficoll-enriched PBMC isolation will be conducted per UNC institutional 
standard procedure. 
• Refer to the LCCC Study Laboratory Manual for details on sample 
collection and processing procedures. 


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7.3.2 Tumor Tissue 
Tumor tissue will be collected at baseline and during study treatment at two time 
points, day 22 and week 16. Collection of tumor tissue at baseline and on day 22 is 
mandatory whereas tumor tissue at week 16 is optional. Tumor tissue specimens as 
well as unstained-blank slides will be stored at UNC-CH TPF and will be sent to 
UNC-CH TPL) for correlative analysis. Details about specific stains are described 
in Section 1.10.2. Refer to the LCCC1620 Study Laboratory Manual for details 
pertaining to the collection, processing, and storage of fresh tumor biopsies, plus 
the procedure for quality control analysis of both archived and freshly isolated 
tumor biopsies.  
7.3.2.1 Research Tissue Collection  
Optional tumor tissue samples from standard of care surgical procedures may 
be collected for research purposes while the subjects are on study. These 
include samples that may have been collected after study enrollment, but prior 
to study biopsies, from any surgical procedure that occurs during study 
enrollment, and those procedures that may occur after study  treatment 
completion as long as the subject is still on study.  
7.3.2.2 Freshly Collected Tumor Tissues 
If archived tumor blocks are not available, subject can still be enrolled to the 
trial on condition that they have fresh tumor biopsy prior to study enrollment. 
 
Subjects must consent to undergo fresh tumor biopsy collection (baseline; day 
22 biopsy mandatory; week 16 is optional). Both tumor biopsies are not 
standard of care and are for research purposes. If palpable tumors from a 
metastatic site are available, then excisional, punch, or core biopsies can be 
performed. If, however, palpable tumors are not available then a core biopsy 
procedure using an 18-gauge needle will be performed by interventional 
radiology. Intrathoracic lesions may be subjected to core biopsy if deemed safe 
by clinical investigator. The number of cores obtained is dependent on the 
subject’s clinical condition at the time of biopsy and the health care 
professional who is performing the procedure. Therefore, it is possible that 
more than one core (up to 5) may be procured for this research project and 
make cores to the one FFPE block.  
 
Freshly collected biopsy cores will be transferred to the UNC-CH TPF and 
will be handled as described in the LCCC1620 Study Laboratory Manual.  
 
 
7.3.3 Handling and Storage of Biospecimens Collected for Correlative Research 
Biospecimens collected as part of this study will be stored in the LCCC TPF (tumor 
tissue) or if needed, in a secure off-site storage facility. All biospecimen samples 
will be obtained in accordance with procedures outlined in the LCCC 1620 Study 
Laboratory Manual and stored in containers with controlled access. Each sample 
will be assigned a unique code number and no identifiable PHI will be on the 


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specimen label. Information about the subject’s disease will be linked to the 
specimens stored in the repository database. TPF-associated research staff, LCCC 
Bioinformatics staff who support the TPF database and the LCCC Data Warehouse, 
and study researchers with IRB-approval for access to PHI for each subject in this 
study will be able to link specimens to relevant medical information. Some results 
from laboratory analyses that occurred during the subject’s participation in the 
clinical study may also be included. This information may be important for 
understanding how the subject’s cancer developed and responded to treatment. 
 
Storage Time 
• 
The biospecimen will be used first and foremost for research purposes outlined 
within the confines of this protocol. Samples will be discarded/destroyed after 
relevant data are collected for this study, unless consent was obtained from the 
subject to use tissue for other research purposes (e.g., TPF consent form was 
signed by the subject). In this circumstance, there is no time limit on how long 
biospecimens may be stored. 
o The investigator must agree to abide by policies and procedures of the TPF 
facility and sign a letter of research agreement for ethical and appropriate 
conduct of their research that utilizes specimens obtained from the TPF 
facility (e.g., use of leftover specimens will require a protocol outlining the 
research plan for biospecimen use).  
 
Compliance Statement 
Biospecimen collection for this study will be conducted in full accordance to all 
applicable UNC-CH Research Policies and Procedures and all applicable Federal 
and state laws and regulations including 45 CFR 46, and the Health Insurance 
Portability and Accountability Act (HIPAA) Privacy Rule. Any episode of 
noncompliance will be documented. 
 
The investigators will perform the study in accordance with this protocol, will 
obtain consent (unless a waiver is granted) and will report unexpected problems in 
accordance with The UNC IRB Policies and Procedures and all federal 
requirements. Collection, recording, and reporting of data will be accurate and will 
ensure the privacy, health, and welfare of research subjects during and after the 
study.  
7.4 
Assessment of Safety  
Any subject who receives treatment on this protocol will be evaluable for toxicity.  
Each subject will be assessed periodically for the development of any toxicity 
according to the Time and Events table. Toxicity will be assessed according to the 
NCI CTCAE version 5.0. Toxicities will be characterized in terms regarding 
seriousness, causality, toxicity grading, and action taken with regard to trial 
treatment.  
 
Progression of the cancer under study is not considered an adverse event unless it 
is considered to be drug related by the investigator. 


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7.5 
Assessment of Efficacy 
Subjects who have received at least 1 dose of a PD-1 inhibitor and the first two 
doses of denosumab will be evaluable for assessment of response and progression.  
Subjects who drop out of the study for any reason (e.g., toxicity of treatment, decide 
to withdraw) will still be followed for PFS and OS.    
7.5.1 Assessment of Disease-Tumor Measurement Based on RECIST v1.1 
Disease assessment according to RECIST v1.1 criteria will include imaging and 
physical examination. Refer to the RECIST v1.1 for additional details 69. To ensure 
comparability between baseline and subsequent disease assessments, the same 
method(s) of assessment (e.g., CT scan, MRI, etc.) will be used throughout the 
study for determining response. Tumor assessment will be completed as outlined 
in the Time and Events table. 
 
Measurable disease will be defined as the presence of at least one measurable lesion 
that can be accurately measured in at least one dimension with the longest diameter 
a minimum size of: 
• >10-mm by CT scan (CT scan slice thickness no greater than 5-mm). 
• 10 mm caliper measurement by clinical exam (lesions which cannot be 
accurately measured with calipers should be recorded as non-
measurable). 
• 20 mm by chest X-ray. 
 
For lymph nodes to be considered pathologically enlarged and measurable, a lymph 
node must be ≥15mm 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.   
 
All other lesions, including small lesions (longest diameter <10-mm or pathological 
lymph nodes with ≥10 to <15-mm short axis) as well as truly non-measurable 
lesions, will be considered non-measurable. Lesions considered truly non-
measurable include: leptomeningeal disease; ascites; pleural/pericardial effusion; 
inflammatory breast disease; lymphangitic involvement of skin or lung, abdominal 
masses/abdominal organomegaly identified by physical exam that is not 
measurable by reproducible imaging techniques. 
 
All measurements should be recorded in metric notation, using calipers, if clinically 
assessed. All baseline evaluations should be performed as close as possible to the 
treatment start 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 lesion at baseline and during follow-up.  Imaging-based 
evaluation should always be done rather than clinical examination unless the 
lesion(s) being followed cannot be imaged but are assessable by clinical exam.  
Clinical lesions will only be considered measurable when they are superficial and 
≥10-mm diameter as assessed using calipers (e.g. skin nodules). In the case of skin 


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lesions, documentation by color photography, including a ruler to estimate the size 
of the lesions, is recommended. 
7.5.2 Baseline Documentation of Target and Non-Target Lesions 
All measurable lesions up to a maximum of 5 (and a maximum of two lesions per 
organ) representative of all involved organs should be identified as target lesions 
and will be recorded and measured at baseline.   
 
Target lesions should be selected on the basis of their size (lesions with the longer 
diameter), be representative of all involved organs, but in addition should be those 
that lend themselves to reproducible repeated measurements.  
 
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, only the short axis is added into the 
sum.  The baseline sum diameters will be used as reference to further characterize 
the objective tumor response of the measurable dimension of the disease. 
 
All other lesions (or sites of disease), including pathological lymph nodes, should 
be identified as non-target lesions and should also be recorded at baseline. 
Measurements are not required and these lesions should be followed as “present” 
or “absent”, or in rare cases “unequivocal progression”.   
7.5.3 Evaluation of Target Lesions using RECIST 1.1 Criteria 
Note: In addition to the information below, also see section 4.3.2 in the international 
criteria proposed by the RECIST v1.1 for special notes on the assessment of target 
lesions69.  
 
Complete Response (CR) − Disappearance of all target lesions.  Any pathological 
lymph node (LN) (whether target or non-target) must have decreased in short axis 
to <10-mm. 
 
Partial Response (PR) − At least a 30% decrease in the sum of the longest 
dimension (LD) of the target lesions taking as reference the baseline sum LD. 
 
Progressive Disease (PD) − At least a 20% increase in the sum of the LD of the 
target lesions taking as reference the smallest sum LD recorded since the treatment 
started, including baseline, 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.  The appearance of one or more new lesions also constitutes PD. 
 
Stable disease (SD) − Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as references the smallest sum LD since the 
treatment started. 


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7.5.4 Evaluation of Non-Target Lesions using RECIST 1.1 Criteria 
Complete Response (CR) − Disappearance of all non-target lesions and 
normalization of tumor marker levels.  All LN must be non-pathological in size 
(<10-mm short axis).   
 
Non-CR)/non-PD − Persistence of one or more non-target lesion(s) or/and 
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. 
7.5.5 Evaluation of Best Overall Response using RECIST v1.1 Criteria 
The best overall response is the best response recorded from the start of the study 
treatment until the end of treatment provided the confirmation criteria are met.  To 
be assigned a status of PR or CR, changes in tumor measurements must be 
confirmed by repeat studies that should be performed > 4 weeks after the criteria 
for response are first met. If a CR/PR cannot be confirmed the original "response" 
should be considered stable disease.  The best overall response will be defined 
according to the following table: 
Overall 
Response First 
Time Point 
Overall Response 
Subsequent Time 
Point 
BEST Overall Response 
CR 
CR 
CR 
CR 
PR 
SD, PD, or PR1 
CR 
SD 
SD provided minimum criteria for SD duration met, 
otherwise, PD 
CR 
PD 
SD provided minimum criteria for SD duration met, 
otherwise, PD 
CR 
NE2 
SD provided minimum criteria for SD duration met, 
otherwise, NE2 
PR 
CR 
PR 
PR 
PR 
PR 
PR 
SD 
SD 
PR 
PD 
SD provided minimum criteria for SD duration met, 
otherwise, PD 
PR 
NE2 
SD provided minimum criteria for SD duration met, 
otherwise, NE2 
NE 
NE2 
NE2 
1 If a CR is truly met at first time point, then any disease seen at a subsequent time point, even disease meeting 
PR criteria relative to baseline, makes the disease PD at that point (since disease must have reappeared after 
CR).  Best response would depend on whether minimum duration for SD was met.  However, sometimes ‘CR’ 
may be claimed when subsequent scans suggest small lesions were likely still present and in fact the subject 
had PR, not CR at the first time point.  Under these circumstances, the original CR should be changed to PR 
and the best response is PR. 2 NE, inevaluable  
7.5.6 Other Efficacy Parameters 
In addition to response rate by RECIST version 1.1 criteria, the study will calculate 
progression-free survival at 6-months and overall survival at 1-year, given that 
these two have proven to be valid early surrogate endpoints of clinical benefit of all 
FDA approved treatments for metastatic melanoma over the last 5 years.   


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8.0 
ADVERSE EVENTS 
8.1 
Definitions  
8.1.1 Adverse Event (AE) 
AE is any untoward medical occurrence (e.g., an abnormal laboratory finding, 
symptom, or disease temporally associated with the use of a drug) in a subject who 
has been administered a pharmaceutical product as part of clinical investigation and 
which does not necessarily have a causal relationship with this treatment.  An AE 
can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of a 
medicinal product, whether or not related to the medicinal product.   
 
Hospitalization for elective surgery or routine clinical procedures that are not the 
result of an AE (e.g., surgical insertion of central line) need not be considered AEs 
and should not be recorded as an AE.  Disease progression should not be recorded 
as an AE, unless it is attributable by the investigator to the study therapy. 
 
From the time of treatment through 30 days following cessation of treatment, all 
AEs must be reported by the investigator. Such events will be recorded at each 
examination on the Adverse Event CRFs. The reporting timeframe for AEs meeting 
any serious criteria is described in section 7.3.3. The investigator will make every 
attempt to follow all subjects with non-serious AEs for outcome. 
8.1.2 Suspected Adverse Reaction (SAR) 
A suspected adverse reaction (SAR) is any AE for which there is a reasonable 
possibility that the drug is the cause. Reasonable possibility means that there is 
evidence to suggest a causal relationship between the drug and the AE. A suspected 
adverse reaction implies a lesser degree of certainty about causality than adverse 
reaction, which means any AE caused by a drug.   
 
Causality assessment to a study drug is a medical judgment made in consideration 
of the following factors: temporal relationship of the AE to study drug exposure, 
known mechanism of action or side effect profile of study treatment, other recent 
or concomitant drug exposures, normal clinical course of the disease under 
investigation, and any other underlying or concurrent medical conditions.  Other 
factors to consider in considering drug as the cause of the AE: 
• Single occurrence of an uncommon event known to be strongly associated with 
drug exposure (e.g., angioedema, hepatic injury, Stevens-Johnson syndrome) 
• One or more occurrences of an event not commonly associated with drug 
exposure, but otherwise uncommon in the population (e.g., tendon rupture); 
often more than once occurrence from one or multiple studies would be needed 
before the sponsor could determine that there is reasonable possibility that the 
drug caused the event.   


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• An aggregate analysis of specific events observed in a clinical trial that 
indicates the events occur more frequently in the drug treatment group than in 
a concurrent or historical control group 
8.1.3 Unexpected AE or SAR 
An AE or SAR is considered unexpected if its specificity or severity is not 
consistent with the applicable product information (e.g., Investigator’s Brochure 
for an unapproved investigational product or package insert/summary of product 
characteristics for an approved product).  Unexpected also refers to AEs or SARs 
that are mentioned in the Investigator’s Brochure as occurring with a class of drugs 
or as anticipated from the pharmacological properties of the drug, but are not 
specifically mentioned as occurring with the particular drug under investigation.   
8.1.4 Serious AE or SAR 
An AE or SAR is considered serious if, in the view of either the investigator or 
sponsor, it results in any of the following outcomes: 
• Death; 
• Is life-threatening (places the subject at immediate risk of death from the event as 
it occurred); 
• Requires inpatient hospitalization (>24 hours) or prolongation of existing 
hospitalization;* 
• Results in congenital anomaly/birth defect; 
• Results in a persistent or significant incapacity or substantial disruption of the 
ability to conduct normal life functions; 
• Important medical events that may not result in death, be life-threatening, or 
require hospitalization may be considered a SAE drug experience when, based 
upon appropriate medical judgment, they may jeopardize the subject and may 
require medical or surgical intervention to prevent one of the outcomes listed in 
the definition.  For reporting purposes, also consider the occurrences of 
pregnancy as an event, which must be reported as an important medical event. 
 
*Hospitalization for anticipated or protocol-specified procedures such as administration of 
chemotherapy, central line insertion, metastasis interventional therapy, resection of primary tumor, or 
elective surgery, will not be considered SAE. 
 
Pregnancy that occurs during the study must also be reported as an SAE. 
 
Note: In addition to the above criteria, AE meeting either of the below criteria, 
although not serious per ICH definition, are reportable to Amgen in the same 
timeframe as SAEs to meet certain local requirements. Therefore, these events are 
considered serious by Amgen or collection purposes. 
● Is a new cancer (that is not a condition of the study); 
● Is associated with an overdose. 


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8.2 
Documentation of non-serious AEs or SARs 
For non-serious AEs or SARs, documentation must begin from day 1 of study 
treatment and continue until the 30-day follow-up period after treatment is 
discontinued.   
 
Collected information should be recorded in the CRF for that subject. Please 
include a description of the event, its severity or toxicity grade, onset and resolved 
dates (if applicable), and the relationship to the study drug. Documentation should 
occur at least monthly.  
8.3 
SAEs or Serious SARs  
8.3.1 Timing 
After informed consent but prior to initiation of study medications, only SAEs 
caused by a protocol-mandated intervention will be collected (e.g. SAEs related to 
invasive procedures such as biopsies, medication washout).  
 
For any other experience or condition that meets the definition of an SAE or a 
serious SAR, recording of the event must begin from day 1 of study treatment and 
continue through the 30-day follow-up period after treatment is discontinued.  
8.3.2 Documentation and Notification 
SAEs or Serious SARs must be recorded in the SAE console within OnCore® for 
that subject within 24 hours of learning of its occurrence.  
8.3.3 Reporting  
IRB Reporting Requirements: 
UNC: 
The UNC-IRB will be notified of all SAEs that qualify as an Unanticipated Problem 
as per the UNC IRB Policies using the IRB’s web-based reporting system within 7 
days of the Investigator becoming aware of the problem.   
 
Affiliate sites:   
For affiliate sites using a local IRB of record, please submit adverse events per local 
IRB policy. 
 
For affiliate sites relying on the UNC-IRB, an aggregated list of all SAEs will be 
submitted to the UNC IRB annually at the time of study renewal according to the 
UNC IRB policies and procedures. In addition, any SAEs that qualify as an 
Unanticipated Problem will be entered into OnCore® by the affiliate site and 
reported to the UNC IRB by the Multicenter Regulatory Associate using the IRB’s 
web-based reporting system within 7 days of the Investigator becoming aware of 
the problem.   
 


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Pregnancy 
Pregnancies and suspected pregnancies (including a positive pregnancy test 
regardless of age or disease state) of a female subject occurring while the subject is 
on study, or within 28 days of the subject’s last dose of study should be recorded 
as SAEs. The subject is to be discontinued immediately from the study.  
 
For Affiliate sites, the pregnancy, suspected pregnancy, or positive pregnancy test 
must be reported to the Multicenter Project Manager immediately (within 24 hours) 
via email (preferred) or facsimile to 919-966-4300.  The Multicenter Project 
Manager will then report the event to the Funding Source (see requirements below).   
 
The female subject should be referred to an obstetrician-gynecologist, preferably 
one experienced in reproductive toxicity for further evaluation and counseling. 
 
The Investigator will follow the female subject until completion of the pregnancy, 
and must document the outcome of the pregnancy (either normal or abnormal 
outcome) and report the condition of the fetus or newborn to the UNC Project 
Manager. If the outcome of the pregnancy was abnormal (e.g., spontaneous or 
therapeutic abortion), the Investigator should report the abnormal outcome as an 
AE. If the abnormal outcome meets any of the serious criteria, it must be reported 
as an SAE.   
 
Amgen Reporting Requirements: 
Please refer to the table below regarding policy for reporting SUSARs, and other 
safety related data to Amgen. 
 
Safety Data 
Timeframe for Submission to AMGEN 
Suspected Unexpected Serious 
Adverse Reaction (SUSARs) 
Sent to AMGEN at time of regulatory 
submission to FDA 
Pregnancy/Lactation 
Within 10 days of Sponsor awareness 
 
To report suspected adverse reactions, contract Amgen at 1-855-465-9442) and 
FDA at 1-800-FDA-1088 or www.fda.gov/medwatch. 
 
Aggregate Reports Required for All Amgen Studies: 
Safety Data 
Timeframe for submission to Amgen 
Annual Safety Report 
(e.g., EU Clinical Trial Directive 
[CTD] Annual Safety Report, and 
US IND Annual Report) 
Annually 
Other Aggregate Analyses 
(any report containing safety data 
generated during the course of a 
study) 
At time of ISS sponsor submission to any  
body governing research conduct (eg, RA, 
IRB, etc) 
Final (End of Study Report, 
including: 
At time of ISS sponsor submission to any 
body governing research conduct (eg, RA, 


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Safety Data 
Timeframe for submission to Amgen 
• Unblinding data for 
blinded studies 
• Reports of unauthorized 
use of a marketed product 
IRB, etc.) but not later than 1 calendar 
year of study completion 
 
Merck Reporting Requirements: 
 
To report suspected adverse reactions, contact Merck Sharp & Dohme Corp., a 
subsidiary of Merck & Co., Inc., at 1-877- 888-4231 and FDA at 1-800-FDA-1088 
or www.fda.gov/medwatch 
8.3.3.1 Events of Clinical Interest 
Selected SAEs are also known as Events of Clinical Interest (ECI)  
 
ECIs for this trial include: 
1. ONJ 
2. Life-threatening hypocalcemia 
3. Serious infection attributed to denosumab 
4. Autoimmune hepatitis or an elevated AST or ALT lab value that is greater than 
or equal to 3 × the ULN and an elevated total bilirubin lab value that is greater 
than or equal to 2 × the ULN and, at the same time, an alkaline phosphatase lab 
value that is <2 × the ULN, as determined by way of protocol-specified 
laboratory testing or unscheduled laboratory testing.* 
 
*Note: These criteria are based upon available regulatory guidance documents. The 
purpose of the criteria is to specify a threshold of abnormal hepatic tests that may 
require an additional evaluation for an underlying etiology. 
 
Although not considered an event of clinical interest, subjects receiving denosumab 
and PD-1 inhibitor in combination may have an increased risk for autoimmune colitis.  
 
 
If an investigator deems that an event is both a serious SAR and unexpected based on the 
extensive knowledge of AE attributed to PD-1 inhibitor alone and denosumab alone (e.g. 
stroke, serious infusion-related reaction, myocardial infarction), it must also (in addition 
to OnCore®) be recorded on the MedWatch Form 3500A, as per 21 CFR 312.32. 
Unexpected AE or adverse reaction refers to an event or reaction that is not listed in the 
investigator’s brochure or is not listed at the specificity or severity that has been 
observed; or if an investigator’s brochure is not required or available, is not consistent 
with the risk information described in the general investigation plan or elsewhere in the 
current IND application.   
 
Process 
If the sponsor deems that an event is both a serious adverse reaction AND unexpected, it 
must also (in addition to OnCore®) be recorded on the MedWatch Form 3500A as per 21 


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CFR 312.32. The MedWatch form should be submitted on MedWatch by the study 
coordinator.  
 
The 
MedWatch 
3500A 
form 
can 
be 
accessed 
at: 
http://www.fda.gov/Safety/MedWatch/HowToReport/DownloadForms/default.htm.  
8.4 
Data and Safety Monitoring Plan 
The Principal Investigator will provide continuous monitoring of subject safety in 
this trial with periodic reporting to the UNC LCCC Data and Safety Monitoring 
Committee (DSMC). As discussed in detail in the Statistical Considerations Section 
8.1, continuous toxicity monitoring will be applied to the incidence of SAE 
attributed to PD-1 inhibitor alone (colitis, hepatitis, hypophysitis, pneumonitis, 
T1DM) as well as to denosumab alone (hypocalcemia, hypophosphatemia, 
osteomyelitis, ONJ). Any trends regarding significantly higher incidence for any of 
these AEs compared to their previously described incidence with either drug alone 
will imply significant interaction between denosumab and PD-1 inhibitor and 
depending on the severity and onset, it may lead to discontinuation of denosumab 
and/or changes in denosumab scheduling. 
 
Meetings/teleconferences will be held at a frequency dependent on study accrual, 
and in consultation with the study Biostatistician (Dr. Anastasia Ivanova).  These 
meetings will include the investigators from all three sites (Drs Moschos, Shirai, 
and Hoimes) as well as protocol nurses, clinical research associates, regulatory 
associates, data managers, and any other relevant personnel the principal 
investigators may deem appropriate.  At these meetings, the research team will 
discuss all issues relevant to study progress, including enrollment, safety, 
regulatory, data collection, etc. 
 
The team will produce summaries or minutes of these meetings. These summaries 
will be available for inspection when requested by any of the regulatory bodies 
charged with the safety of human subjects and the integrity of data including, but 
not limited to, the oversight Office of Human Research Ethics (OHRE) Biomedical 
IRB, the Oncology Protocol Review Committee (PRC) or the North Carolina 
TraCS Institute Data and Safety Monitoring Board (DSMB).   
 
The UNC LCCC Data and Safety Monitoring Committee (DSMC) will review the 
study on a regular (quarterly to annually) basis, with the frequency of review based 
on risk and complexity as determined by the UNC Protocol Review Committee. 
The UNC PI will be responsible for submitting the following information for 
review: 1) safety and accrual data, including the number of subjects treated; 2) 
significant developments reported in the literature that may affect the safety of 
participants or the ethics of the study; 3) preliminary response data; and 4) 
summaries of team meetings that have occurred since the last report.  Findings of 
the DSMC review will be disseminated by memo to the UNC PI, PRC, and the 
UNC IRB and DSMB. 


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9.0 
STATISTICAL CONSIDERATIONS 
This is a phase II study of two FDA-approved drugs, one of which is standard of 
care for metastatic melanoma (e.g. pembrolizumab or nivolumab) whereas the other 
is FDA-approved for a specific condition (e.g. bone metastases), irrespective of the 
cancer type. We believe that a dedicated phase I study is not required and that a 
phase II study using those two drugs at the FDA-approved doses will be able to 
systematically investigate both safety and efficacy of this treatment combination.  
9.1 
Study Design/Study Endpoints 
All subjects will receive nivolumab (or pembrolizumab) plus denosumab for up to 
year. Drugs will be sequentially introduced, such that correlative analysis can be 
performed to investigate the mechanistic (direct antitumor vs. indirect 
immunomodulatory effect) and pharmacodynamic (target tissue saturation studies) 
effect of denosumab alone and in combination with nivolumab (or pembrolizumab) 
by performing serial peripheral blood collections (up to 5 timepoints) and tumor 
collection (up to 3 time points). Assessment of response to the treatment 
combination will be assessed at week 16 and then every 12 weeks of nivolumab (or 
pembrolizumab) treatment for up to 1-year.  
9.1.1 Definitions 
Evaluable subjects are those who complete all activities by at least day 22, namely: 
(a) they have received the two denosumab injections on day 1 and day 8, (b) they 
have received the first nivolumab (or pembrolizumab) infusion on day 22, and (c) 
they have completed day 22 mandatory tumor biopsy. The rationale for this is to 
answer the co-primary translational endpoints regarding denosumab-alone 
immunomodulatory effects in peripheral blood and tumor tissue with sufficient 
power versus denosumab and nivolumab (or pembrolizumab) in combination. 
Accruing the projected number of subjects along with baseline and day 22 
peripheral blood and tumor tissue endpoints is considered one of the “translational 
successes” of this study. 
SAEs are grade 3 or higher AE, irrespective of attribution to study drug(s). 
PFS is measured from the date of enrollment on study to the date of documented 
progression. The rate of subjects who are progression-free will be specifically 
calculated at 6 months from study entry. 
OS is measured from the date of enrollment on study to the recorded date of death. 
The rate of subjects who are alive at 1-year will be specifically calculated for the 
study. 
9.1.2 Correlative Studies 
This study is unusual for “traditional” phase II cancer trials in that the primary 
endpoints are translational correlatives and not a clinical efficacy endpoint. 
Correlative analysis relies on the collection of peripheral blood and tumor tissue on 
which power size calculations are based (see Section 8.2, Sample Size and 


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Accrual). Specific data analysis plans for the correlative studies are presented in 
Section 8.3. 
9.1.3 Toxicity Assessment 
Toxicity assessment is an important secondary endpoint, because toxicity from 
concurrent administration of these two drugs has not been systematically addressed, 
despite the fact that denosumab and nivolumab (or pembrolizumab) are frequently 
co-administered in community medical oncology practice, in particular for subjects 
with bone metastases from metastatic melanoma. Given the presumed mechanism 
of action of denosumab, namely increased thymic output of autoreactive T cells, 
we cannot exclude the possibility that the incidence of several immune-mediated 
SAE attributed to nivolumab (or pembrolizumab) may actually be increased due to 
concurrent administration of denosumab. We will therefore apply continuous 
toxicity monitoring focusing on the incidence of SAE attributed to nivolumab (or 
pembrolizumab) and with toxicity boundary rules that should not by any chance 
occur more frequently that the well-established immune-mediated effects from the 
concurrent ipilimumab-nivolumab administration. More specifically, from the 
large phase III study of pembrolizumab versus ipilimumab in subjects with 
metastatic melanoma the most significant SAEs have been colitis (2.5%), hepatitis 
(1.8%), hypophysitis (0.4%), pneumonitis (0.4%), type I diabetes mellitus (0.4%)10. 
In the phase III study of subjects with metastatic melanoma who were randomized 
to nivolumab alone, versus ipilimumab alone, versus nivolumab plus ipilimumab, 
the following frequent SAE were observed in the concurrent ipilimumab-
nivolumab arm. Diarrhea (9.3%), fatigue (4.2%), rash (4.8%), ALT increase 
(8.3%), AST increase (6.1%), colitis (7.7%). In addition, a total of 29.4% patients 
who were treated with ipilimumab-nivolumab were discontinued from the study 
due to treatment-related AE 9. If the incidence of any of the above stated SAE that 
were observed in the ipilimumab-nivolumab trial is reached at any point in our trial, 
we will consider that the denosumab and PD-1 inhibitor combination is toxic, and 
we will therefore discontinue denosumab administration; instead patients will only 
be treated with PD-1 inhibitor alone. Trial can also permanently stop at any point 
during the study, if there is evidence that the probability for a patient of prematurely 
stopping denosumab due to treatment-related AEs is 30% or higher.  
 
In the large phase II study of denosumab alone in patients with giant cell tumor of 
the bone the incidence of SAE attributed to denosumab alone was 0% for grade ≥3 
hypocalcemia, 3% for hypophosphatemia, 1% for osteomyelitis, and 1% for 
osteonecrosis of the jaw 70. These four AEs will be monitored throughout our study 
and toxicity stopping rules will apply. Denosumab alone can be discontinued within 
a particular patient if such SAE attributed to denosumab alone may occur and 
cannot be treated with calcium supplementation. However, denosumab (and 
therefore the study overall) can prematurely stop if the incidence of any of the four 
stated SAE attributed to denosumab exceeds 10%.  In addition, these four distinct 
SAEs attributed to denosumab will be monitored throughout the study.  
 




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9.2 
Sample Size and Accrual 
Our goal is to enroll 25 evaluable subjects; therefore, we expect to enroll about 28 
subjects. The immune-mediated mechanism of action of denosumab alone (co-
primary translational endpoints) versus the combination of denosumab and 
nivolumab (or pembrolizumab) will be evaluated in blood using multiparameter 
flow cytometry and ELISA assays and in tumor samples through IHC and IF. Blood 
samples will be collected at baseline (week 0) and at weeks 3, 16, 28 and 40. Tumor 
samples will be collected at baseline and weeks 3 and 16. For each subject and for 
each of these measurements, we will compute the difference from the 
corresponding baseline, week 0 for denosumab alone and week 3 for the 
combination of denosumab and nivolumab (or pembrolizumab). The differences 
will be tested with one-sample t-tests at one-sided 0.05 level each. No adjustment 
for multiple testing will be performed. With the sample size of 25 we will be able 
to detect a significant change from baseline with power 80% if the true effect size 
is 0.51. This is in line with previously published numbers 65,72-74.  
9.3 
Data Analysis Plans 
For each assay in the serum or peripheral blood there will be between 3 to 5 time 
points (baseline, day 22, week 16, week 28, week 40), depending on whether 
subjects continue to have clinical benefit to the denosumab-anti-PD-1 agent 
combination beyond the week 16 time-point of first assessment of antitumor and 
throughout the year-long duration of the study. Descriptive statistics will be 
applied. We will plot means and standard errors for each time point; logarithmic or 
other transformation may be applied for data normalization, if necessary. Particular 
emphasis will be put on assessment of changes attributed to denosumab alone 
(comparison between the day 22 and baseline) as well as changes attributed to 
denosumab-anti-PD-1 agent combination (comparison between the week 16 and 
baseline). Serum measurements of free RANKL and OPG levels are continuous 
variables (from zero to infinity, in mol/L) whereas peripheral blood immune cell 
subsets described in Section 1.10.1.2 (items 1, 2, 5, 6, 7, and 8) are percentages of 
cell subset (from 0 to 100). CD4+ and CD8+ sjTREC (item 3 in Section 1.10.1.2) 
are continuous variables (from zero to infinity in number of cells per 5 x 107 
PBMCs). All the above analyses will be performed by Dr. Anastasia Ivanova.  
 
TCR repertoire analysis will be performed by Dr. Benjamin Vincent, whose 
laboratory has developed and published methods for assessment of clonality and 
diversity of the immune receptor repertoire in cancer 75. 
 
Tumor tissues will be collected at baseline and day 22. Week 16 tumor tissue 
collection is optional. For all tumor tissue studies, which are described at length in 
section 1.10.2, quantitative computer imaging analysis will be applied using 
commercially available computer imaging software (Definiens Tissue Studio and 
Visiopharma). Descriptive statistics will be applied. We will plot means and 
standard errors for each time point; logarithmic or other transformation may be 
applied for data normalization, if necessary. The differences from corresponding 


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baseline for each measurement and at for each time point will be tested with one-
sample t-tests at one-sided 0.05 level each. 
 
AEs of the denosumab-anti-PD-1 agent combination will be tabulated and 95% 
confidence interval will be computed. The overall RR (CR + PR) at 16 weeks will 
be estimated as a proportion of subjects with RR and 95% confidence interval will 
be computed. PFS at 6 months and OS at 1 year will be estimated using Kaplan-
Meier method.  
 


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10.0 STUDY MANAGEMENT 
10.1 
Institutional Review Board (IRB) Approval and Consent 
It is expected that the IRB will have the proper representation and function in accordance 
with federally mandated regulations.  The IRB should approve the consent form and 
protocol. 
 
In obtaining and documenting informed consent, the investigator should comply with the 
applicable regulatory requirement(s), and should adhere to Good Clinical Practice (GCP) 
and to ethical principles that have their origin in the Declaration of Helsinki. 
 
 
 
Before recruitment and enrollment onto this study, the subject will be given a full 
explanation of the study and will be given the opportunity to review the consent form. Each 
consent form must include all the relevant elements currently required by the FDA 
Regulations and local or state regulations. Once this essential information has been 
provided to the subject and the investigator is assured that the subject understands the 
implications of participating in the study, the subject will be asked to give consent to 
participate in the study by signing an IRB-approved consent form. 
 
Prior to a subject’s participation in the trial, the written informed consent form should be 
signed and personally dated by the subject and by the person who conducted the informed 
consent discussion. 
10.2 
Required Documentation 
Before the study can be initiated at any site, the following documentation must be provided 
to the CPO at UNC-CH. 
 
• A copy of the official IRB approval letter for the protocol and informed consent. 
• IRB membership list. 
• CVs and medical licensure for the principal investigator and any sub-
investigators who will be involved in the study. 
• Financial Disclosures 
• Form FDA 1572 appropriately filled out and signed with appropriate 
documentation  
• CAP and CLIA Laboratory certification numbers and institution lab normal 
values. 
• Executed clinical research contract. 
10.3 
Registration Procedures 
All subjects must be registered with the LCCC CPO Multicenter Office at the University 
of North Carolina before enrollment to study. To register a subject call the Multicenter 
office at 919-966-7359 Monday-Friday 8:30 am – 5:00 pm EST. Scan and email the UNC 
Project Manager (CPOMultiCenter@med.unc.edu; preferred) or fax (919-966-4300) the 
registration form, signed informed consents, signed eligibility form and all source 


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documents to confirm eligibility. When sending registration request with eligibility 
documentation, please allow 24 hours for source to be reviewed. 
 
10.4 
Data Management and Monitoring/Auditing 
The CPO Multicenter Office of the UNC LCCC will serve as the coordinating center for 
this trial. Data will be collected through a web based clinical research platform, OnCore®.  
Other study institutions will be given a password to directly enter their own data onto the 
web site via electronic case report forms (eCRFs).  Multicenter personnel will coordinate 
and manage data for quality control assurance and integrity. 
 
All data will be collected and entered into OnCore® by the affiliate study teams at 
participating institutions.  The investigators at each site will allow monitors to review all 
source documents supporting data entered into OnCore®. The Multicenter Data 
Coordinator can be reached at 919-843-2742 or 1-877-668-0683. 
 
All data will be monitored and source data will be verified on selected subjects.  Queries 
will be issued on an ongoing basis on all subjects. Participating sites should respond to data 
queries within 14 days of receipt. The LCCC compliance committee or their designee will 
audit trial sites every twelve months while still enrolling or subjects are still on treatment. 
Participating sites must send source and regulatory documents to LCCC upon request, for 
remote monitoring and/or audit review. 
10.5 
Adherence to the Protocol 
Except for an emergency situation in which proper care for the protection, safety, and well-
being of the study subject requires alternative treatment, the study shall be conducted 
exactly as described in the approved protocol.   
10.5.1 Emergency Modifications 
UNC and affiliate investigators may implement a deviation from, or a change of, the 
protocol to eliminate an immediate hazard(s) to trial subjects without prior UNC or their 
respective institution’s IRB/IEC approval/favorable opinion.   
 
For any such emergency modification implemented, a UNC-CH IRB modification form 
must be completed by UNC Research Personnel within five (5) business days of making 
the change.   
 
For Institutions Relying on UNC’s IRB: 
For any such emergency modification implemented, a UNC IRB modification form must 
be completed by UNC Research Personnel within five (5) business days of making the 
change.   
 
For Institutions Relying on Their Own IRB: 
For Affiliate investigators relying on their own institution’s IRB, as soon as possible after 
the modification has been made, the implemented deviation or change and the reasons for 
it should be submitted to: 


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• To UNC Principal Investigator for agreement  
• The Affiliate institution’s IRB for review and approval.  (Once IRB’s 
response is received, this should be forwarded to the Multicenter Regulatory 
Associate). 
10.5.2 Single Subject Exceptions 
Eligibility single subject exceptions are not permitted for Lineberger Comprehensive 
Cancer Center Investigator Initiated Trials under any circumstances. Other types of single 
subject exceptions may be allowed if proper regulatory review has been completed in 
accordance with Lineberger Comprehensive Cancer Center’s Single Subject Exceptions 
Policy.  
10.5.3 Other Protocol Deviations/Violations 
According to UNC’s IRB, a protocol deviation is any unplanned variance from an IRB 
approved protocol that:  
• Is generally noted or recognized after it occurs 
• Has no substantive effect on the risks to research participants 
• Has no substantive effect on the scientific integrity of the research plan or the 
value of the data collected  
• Did not result from willful or knowing misconduct on the part of the 
investigator(s).  
 
An unplanned protocol variance is considered a violation if the variance meets any of the 
following criteria:  
• Has harmed or increased the risk of harm to one or more research participants. 
• Has damaged the scientific integrity of the data collected for the study. 
• Results from willful or knowing misconduct on the part of the investigator(s). 
• Demonstrates serious or continuing noncompliance with federal regulations, 
State laws, or University policies. 
 
If a deviation or violation occurs, please follow the guidelines below. 
 
For Institutions Relying on UNC’s IRB: 
Protocol Deviations: UNC or Affiliate personnel will record the deviation in OnCore®, 
and report to any sponsor or data and safety monitoring committee in accordance with their 
policies.  Deviations should be summarized and reported to the IRB at the time of 
continuing review. 
 
Protocol Violations: Violations should be reported by UNC personnel within one (1) week 
of the investigator becoming aware of the event using the same IRB online mechanism 
used to report Unanticipated Problems.   
 
For Institutions Relying on Their Own IRB: 
In addition to adhering to the policies regarding protocol compliance set forth by your 
institution’s IRB, the following is also required: 


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Protocol Deviations: In the event a deviation from protocol procedures is identified, 
record the deviation in OnCore®. 
 
Protocol Violations: Any protocol violation that occurs must be reported to your IRB per 
institutional policies and reported to the UNC Multicenter Project Manager within 5 days. 
UNC will determine if the violation affects the safety of the subject and integrity of the 
data.  Once your institution’s IRB response is received, please forward to the Multicenter 
Regulatory Associate. 
 
Unanticipated Problems: 
Any events that meet the criteria for “Unanticipated Problems” as defined by UNC’s IRB 
must be reported by the Study Coordinator using the IRB’s web-based reporting system.   
 
Affiliate Sites: 
Any events that meet the criteria for “Unanticipated Problems (UPs)” as defined by UNC’s 
IRB must also be reported to the UNC Multicenter Project Manager. The Multicenter 
Regulatory Associate will report the event to the UNC IRB using the IRB’s web-based 
reporting system.  Examples of such UPs include a lost or stolen laptop computer that 
contains sensitive study information. 
10.6 
Amendments to the Protocol 
Should amendments to the protocol be required, the amendments will be originated and 
documented by the Principal Investigator at UNC-CH. It should also be noted that when 
an amendment to the protocol substantially alters the study design or the potential risk to 
the subject, a revised consent form might be required.   
 
For Institutions Relying on UNC’s IRB: 
The written amendment, and if required the amended consent form, must be sent to UNC-
CH’s IRB for approval prior to implementation.   
 
For Institutions Relying on Their Own IRB: 
Investigators must submit the amendment to their institution’s IRB for approval.  For 
multicenter studies, any multicenter site must submit their informed consent revisions to 
the Multicenter Regulatory Associate prior to submission to their IRB.   
.   
10.7 
Record Retention 
Study documentation includes all eCRFs, data correction forms or queries, source 
documents, Sponsor-Investigator correspondence, monitoring logs/letters, and regulatory 
documents (e.g., protocol and amendments, IRB correspondence and approval, signed 
subject consent forms). 
 
Source documents include all recordings of observations or notations of clinical activities 
and all reports and records necessary for the evaluation and reconstruction of the clinical 
research study. 


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Government agency regulations and directives require that all study documentation 
pertaining to the conduct of a clinical trial must be retained by the Principal Investigator.  
In the case of a study with a drug seeking regulatory approval and marketing, these 
documents shall be retained for at least two years after the last approval of marketing 
application in an International Conference on Harmonization (ICH) region.  In all other 
cases, study documents should be kept on file until three years after the completion and 
final study report of this investigational study. 
10.8 
Obligations of Investigators 
The Principal Investigator is responsible for the conduct of the clinical trial at the site in 
accordance with Title 21 of the Code of Federal Regulations and/or the Declaration of 
Helsinki.  The Principal Investigator is responsible for personally overseeing the treatment 
of all study subjects. The Principal Investigator must assure that all study site personnel, 
including sub-investigators and other study staff members, adhere to the study protocol and 
all FDA/GCP/NCI regulations and guidelines regarding clinical trials both during and after 
study completion. 
 
The Principal Investigator at each institution or site will be responsible for assuring that all 
the required data will be collected and entered into the eCRFs. Periodically, monitoring 
visits will be conducted, and the Principal Investigator will provide access to his/her 
original records to permit verification of proper entry of data. At the completion of the 
study, all eCRFs will be reviewed by the Principal Investigator and will require his/her 
final signature to verify the accuracy of the data. 
 


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Smyth MJ, Yagita H, McArthur GA: Combination Anti-CTLA-4 and Anti-
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55. 
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72. 
Moschos SJ, Edington HD, Land SR, et al: Neoadjuvant treatment of regional 
stage IIIB melanoma with high-dose interferon alfa-2b induces objective tumor regression in 


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association with modulation of tumor infiltrating host cellular immune responses. J Clin Oncol 
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12.0 APPENDICES 
12.1 
Appendix A. ECOG Performance Status 
 
Grade 
Description 
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. Capable of only limited self-care, 
confined to bed or chair 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. 
* As published in Am. J. Clin. Oncol.: Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, 
T.E., McFadden, E.T., Carbone, P.P. Toxicity and Response Criteria of the Eastern Cooperative 
Oncology Group. Am J Clin Oncol 5:649-655, 1982. The Eastern Cooperative Oncology Group, 
Robert Comis M.D., Group Chair. 
 
 
 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
95 
12.2 
Appendix B. Time and Events Table (For Subjects Receiving Pembrolizumab) 
(Note: Week 1 includes days 1 through 7; week 2 includes days 8 through 14; week 3 includes days 15 through 21, etc.) 
Assessments1 
Pre-
Study1 
Study Treatment (cycle 1 = 21 days) 
End of 
Tx 
Long-
term 
follow 
up21 
C1D1 
C1D8 
C2D1 
C3D1 
C3D15 
C4D1 
C5D1 
C6D1 
C7D1 –  
C17D1 
Day-
21 to -
0 
Wk1 
D1±1d 
Wk2 
D8±1d 
Wk4 
D22±2d 
Wk7 
D43±3d 
Wk9 
D56±7d 
Wk10 
D64±7d 
Wk13 
D85±7d 
Wk16 
D106±7d 
*Wks17-4920 
(Wks22/28/34/ 
40/46±2wks) 
Wk52 
Informed Consent 
× 
 
 
 
 
 
 
 
 
 
 
 
History, PE2 
× 
× 
 
× 
× 
 
× 
 
× 
× 
× 
× 
Serum Pregnancy Test 
×3 
 
 
 
 
 
 
 
 
 
 
 
ECOG Performance Status 
× 
×4 
 
× 
× 
 
× 
 
× 
× 
× 
 
Concomitant meds review 
× 
× 
 
Throughout the study 
× 
 
CBC with differential5  
× 
×4 
 
× 
× 
 
× 
 
× 
× 
× 
 
Serum chemistries6 
× 
×4,6 
 
× 
× 
 
× 
× 
× 
× 
× 
 
Ca+, Mg+, phosphorus7 
 
 
× 
 
 
× 
 
 
 
×20 
 
 
Endocrine tests8 
× 
 
 
 
 
 
× 
 
× 
×20 
× 
 
Urinalysis 
× 
×4 
 
 
 
 
 
 
× 
× 
× 
 
Toxicity assessment9 
× 
× 
× 
× 
× 
× 
× 
× 
× 
× 
× 
 
Brain MRI10 
× 
 
 
 
 
 
 
 
 
 
 
 
Whole body CT11 
× 
 
 
 
 
 
 
 
× 
×11 
×11 
 
Denosumab12 
 
× 
× 
× 
 
× 
 
× 
× 
×20 
 
 
Pembrolizumab13 
 
 
 
× 
× 
 
× 
× 
× 
×13, 20 
×14 
 
Peripheral blood mononuclear 
cells15 
 
× 
 
× 
 
 
 
 
× 
×15,20 
× 
 
Archival tissue (baseline) / 
Tumor Biopsies16 
×17 
 
 
×18 
 
 
 
 
×19 
 
 
 
Dental examination 
× 
 
 
 
 
 
 
 
 
 
 
 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
96 
Footnotes to Time and Events Table 
 
1. All screening labs to be obtained within 72 hours prior to registration. All study visits should be 
performed within ± 1 day for the first two weeks of study therapy. For the Day 21 visit the window is ± 
2 days, then ± 3 days for assessments scheduled on Day 28 (week 5) through Day 42 (Week 7) and then 
± 7 days for all subsequent visits. 
2. Complete history/physical at baseline only, thereafter focused history/physical exam on 
symptoms/toxicity.  
3. Serum pregnancy test should be performed within 72 hours prior to the first dose of denosumab 
4. All screening labs to be obtained within 72 hours prior to registration.    
5. Will be performed at baseline and every other pembrolizumab infusion starting from day 21, as per 
standard of care. 
6. Include sodium, potassium, chloride, CO2, BUN, creatinine, glucose, calcium, albumin, total protein, 
total bilirubin, AST, ALT, alkaline phosphatase, magnesium, phosphorus, LDH, amylase and lipase. All 
listed serum chemistries will be performed before each pembrolizumab infusion as per standard of care.  
7. On the days of denosumab injections that do not coincide with pembrolizumab infusions, calcium, 
magnesium, and phosphorus will be performed immediately before denosumab injections. 
8. Include cortisol levels, free T4 (fT4)/TSH, prolactin (women only), and testosterone (males only). 
9. Toxicity assessments per NCI-CTCAE version 5.0. 
10. Is performed at baseline; on future visits only if clinically indicated, as per standard of care. 
11. Includes CT of the neck (if applicable), chest, abdomen, pelvis with IV contrast. Scans are performed at 
baseline, week 16, and then every 12 weeks thereafter (i.e., weeks 28, 40, and 52).  Scans may be 
performed within ±7 days of the scheduled visit. 
12. Denosumab will be administered s.c. as a flat dose, 120 mg every 4 weeks, as per FDA-approved dose, 
for up to a year until intolerable toxicity or disease progression occurs. An extra 120 mg loading dose of 
denosumab will be administered on day 8. See Dosing schema in section 4.2.1. Denosumab will not be 
continued beyond the one-year duration of the trial, irrespective of treatment response. 
13. Pembrolizumab will be administered every 3 weeks, 200 mg, for up to a year, as per FDA-approved dose 
starting on D21 of the study until intolerable toxicity or disease progression occurs.  See Dosing schema 
in section 4.2.1. 
14. Following completion of the trial at 1 year and on condition that there is no disease progression, 
pembrolizumab can be continued at the opinion of the investigator, as per standard of care guidelines. 
15. PBMCs will be isolated from peripheral blood (approximately 100 mL at each time point) using the 
Ficoll isolation technique; an aliquot will be used for multiparameter flow cytometry, another for 
immune receptor repertoire analysis, and another for sjTCR excision cycle analysis. Samples will be 
collected at baseline on D1/wk1 and on D21/wk4, D106/wk16, wk28, and wk40. 
16. Archival tissue or fresh tumor will be collected at baseline and a mandatory excisional biopsy will be 
performed on D21 of the study. An optional excisional biopsy should be obtained on D106 (week 16).  
Tumor tissue will be submitted to the Tissue Pathology Lab (TPL) for IHC and multicolor IF. 
17. If archived tumor blocks are not available, subject can still be enrolled on condition that he/she has a 
tumor biopsy prior to study enrollment. 


LCCC 1620 
 
CONFIDENTIAL 
PI: Stergios Moschos, MD 
 
UNIVERSITY OF NORTH CAROLINA 
Amendment 4 Version 1.1 
 
 July 7, 2022 
97 
18. Mandatory (excisional, punch or CT-guided). 
19. Optional. 
20. Following radiographic disease assessment on week 16, subjects will be seen by the investigator on 
every other pembrolizumab infusion (i.e., 6 weeks on D1 of weeks 22, 28, 34, 40, 46 and at the end of 
treatment visit at week 52) for safety assessment and on every fourth pembrolizumab infusion (i.e., 12 
weeks) for response assessment for up to a year. See sections identified below for details of scheduled 
assessments. 
21. Subjects who have not progressed on pembrolizumab plus denosumab after a year of treatment will be 
followed every 3 months for up to 2 years (i.e. a total of 3 years since study initiation) with at least phone 
calls. The following information will be recorded: (a) details regarding systemic treatment for their 
melanoma that they are currently on (type, response if any, duration on treatment, treatment-related side 
effects), (b) whether they have progressed (and when) from current PD-1 inhibitor treatment, (c) whether 
treatment-related adverse events that subjects may have developed during the year of denosumab-
pembrolizumab treatment have resolved, and when, (d) whether they are alive or not; if dead, precise 
date of death. Subjects who have progressed on pembrolizumab plus denosumab within the year of the 
study duration will be followed every 3 months for up to 3 years since study initiation with at least phone 
calls. The following information will be recorded: (a) systemic treatment for their melanoma they are 
currently on (type, response if any, duration on treatment, treatment-related side effects), (b) whether 
treatment-related AEs that subjects may have developed during the year of denosumab-pembrolizumab 
treatment have resolved, and when, (c) whether they are alive or not; if dead, precise date of death