A Phase II Study Assessing the Effect of Pembrolizumab Induced Changes to the NK Cell Exhaustion Phenotype on the Efficacy of PD-1 Targeted Treatment in Patients With Unresectable Stage III or Stage IV Melanoma
Phase: PHASE2
Conditions: Melanoma
Sponsor: Nina Bhardwaj

A Phase II Study Assessing the Effect of Pembrolizumab Induced Changes to the NK Cell 
Exhaustion Phenotype on the Efficacy of PD-1 Targeted Treatment in Patients With 
Unresectable Stage III or Stage IV Melanoma   
PI: Dr. Nina Bhardwaj 
NCT03241927 
Document Date: June 23, 2017 

Product: Pembrolizumab 
Protocol/Amendment No.: 01/ 07 
 
 
 
 
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SPONSOR: Icahn School of Medicine at Mt Sinai  
 
TITLE: A phase II Study Assessing the Effect of Pembrolizumab Induced Changes to 
the NK Cell Exhaustion Phenotype on the Efficacy of PD-1 Targeted Treatment in 
Patients with Unresectable Stage III or Stage IV Melanoma 
 
Principal Investigator:  Nina Bhardwaj 
Co-Principal Investigator: Philip Friedlander 
 
IND NUMBER: 130351  
 
Original protocol: December 15, 2015 
Amendment 1: February 19, 2016 
Amendment 2: June 20, 2016 
Amendment 3: August 10, 2016 
Amendment 4: September 13, 2016 
Amendment 5: December 16, 2016 
Amendment 6: January 23, 2017 
Amendment 7: June 23, 2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
1.0 
TRIAL SUMMARY  
 
 
 
 
 
 
 
6 
2.0 
TRIAL DESIGN 
 
 
 
 
 
 
 
 
6 
2.1 Trial Design 
 
 
 
 
 
 
 
 
6 
2.2 Trial Diagram 
 
 
 
 
 
 
 
 
7 
3.0 
OBJECTIVES & HYPOTHESES  
 
 
 
 
 
8 
3.1 Primary Objective & Hypothesis 
 
 
 
 
 
8 
3.2 Secondary Objectives & Hypotheses  
 
 
 
 
8 
4.0  
BACKGROUND & RATIONALE  
 
 
 
 
 
9 
4.1 Background 
 
 
 
 
 
 
 
 
9 
 
4.1.1 Melanoma 
 
 
 
 
 
 
 
 
9 
 
4.1.2 NK cells  
 
 
 
 
 
 
 
 
10
 
 
Preliminary data  
 
 
 
 
 
 
 
10 
4.1.3 Pharmaceutical and Therapeutic Background (pembrolizumab) 
 
14 
4.1.4 Preclinical and Clinical Trial Data 
 
 
 
 
 
15 
 
4.2 Rationale  
 
 
 
 
 
 
 
 
15 

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4.2.1 Rationale for the Trial and Selected Subject Population 
 
 
16 
4.2.2 Rationale for Dose Selection/Regimen/Modification   
 
 
16 
5.0  
METHODOLOGY  
 
 
 
 
 
 
 
17 
 
 5.1 Entry Criteria 
 
 
 
 
 
 
 
 
17 
5.1.1 Melanoma Subject Inclusion Criteria  
 
 
 
 
17 
5.1.2 Melanoma Subject Exclusion Criteria  
 
 
 
 
19 
5.1.3 Healthy subject inclusion and exclusion criteria  
 
 
 
21 
5.2 Trial Treatments  
 
 
 
 
 
 
 
21 
 
5.2.1 Dose Selection/Modification 
 
 
 
 
 
 
21 
 
5.2.1.1 Dose Selection 
 
 
 
 
 
 
 
21 
5.2.1.2 Dose Modification  
 
 
 
 
 
 
 
22 
5.2.2 Timing of Dose Administration  
 
 
 
 
 
23 
5.2.3 Trial Blinding/Masking  
 
 
 
 
 
 
23 
 
5.3 Randomization or Treatment Allocation 
 
 
 
 
23 
5.4 Concomitant Medications/Vaccinations (allowed & prohibited) 
 
23 
5.4.1 Acceptable Concomitant Medications  
 
 
 
 
24 
5.4.2 Prohibited Concomitant Medications  
 
 
 
 
24 
5.5 Rescue Medications & Supportive Care 
 
 
 
 
25 
5.5.1 Supportive Care Guidelines 
 
 
 
 
 
 
25 
5.6 Diet/Activity/Other Considerations 
 
 
 
 
 
28 
5.6.1 Diet 
 
 
 
 
 
 
 
 
 
28 

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5.6.2 Contraception 
 
 
 
 
 
 
 
 
28 
5.6.3 Use in Pregnancy 
 
 
 
 
 
 
 
29 
5.6.4 Use in Nursing Women  
 
 
 
 
 
 
29 
5.7 Subject Withdrawal/Discontinuation Criteria  
 
 
 
30 
5.7.1 Discontinuation of Study Therapy after CR  
 
 
 
 
31 
5.8 Subject Replacement Strategy  
 
 
 
 
 
31 
5.9 Clinical Criteria for Early Trial Termination  
 
 
 
31 
6.0  
TRIAL FLOW CHART 
 
 
 
 
 
 
 
32 
6.1 Study Flow Chart 
 
 
 
 
 
 
 
32 
7.0  
TRIAL PROCEDURES 
 
 
 
 
 
 
 
35 
7.1 Trial Procedures  
 
 
 
 
 
 
 
35 
 
7.1.1 Administrative Procedures 
 
 
 
 
 
 
35 
7.1.2 Clinical Procedures/Assessments 
 
 
 
 
 
37 
7.1.3 Laboratory Procedures/Assessments 
 
 
 
 
 
41 
7.1.4 Visit Requirements 
 
 
 
 
 
 
 
43 
7.2 Assessing and Recording Adverse Events 
 
 
 
 
44 
7.2.1 Definition of an Overdose for This Protocol and Reporting of Overdose to the 
Sponsor and to Merck  
 
 
 
 
 
 
45 
7.2.2 Reporting of Pregnancy and Lactation to the Sponsor and to Merck  
45 
7.2.3 Immediate Reporting of Adverse Events to the Sponsor and to Merck 
46 
7.2.4 Evaluating Adverse Events 
 
 
 
 
 
 
48 
7.2.5 Sponsor Responsibility for Reporting Adverse Events  
 
 
51 
8.0  
STATISTICAL ANALYSIS PLAN 
 
 
 
 
 
51 
 
8.1 Primary Objective 
 
 
 
 
 
 
 
51 
 
8.2 Secondary Endpoints 
 
 
 
 
 
 
 
52 
 
8.3 Stopping Rules  
 
 
 
 
 
 
 
52 

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9.0 
LABELING, PACKAGING, STORAGE AND RETURN OF CLINICAL 
SUPPLIES 
 
 
 
 
 
 
 
 
 
53 
 
9.1 Investigational Product   
 
 
 
 
 
 
53 
9.2 Packaging and Labeling Information  
 
 
 
 
53 
9.3 Clinical Supplies Disclosure 
 
 
 
 
 
 
53 
9.4 Storage and Handling Requirements  
 
 
 
 
53 
9.5 Returns and Reconciliation 
 
 
 
 
 
 
54 
10.0  ADMINISTRATIVE AND REGULATORY DETAILS  
 
 
54 
10.1 Compliance 
 
 
 
 
 
 
 
 
54 
10.2 Compliance with Trial Registration and Results Posting Requirements 56 
10.3 Quality Management System  
 
 
 
 
 
56 
11.0  APPENDICES 
 
 
 
 
 
 
 
 
57 
11.1  ECOG Performance Status 
 
 
 
 
 
 
57 
11.2  RECIST Evaluation criteria  
 
 
 
 
 
57 
11.3  Common Terminology Criteria for Adverse Events V4.0 (CTCAE) 
58 
11.4 Response Evaluation Criteria according to Immune-Related Response 
Criteria – iRC  
 
 
 
 
 
 
 
58 
11.5  Events of Clinical Interest Guidance Document 
 
 
 
58 
12.0 REFERENCES 
 
 
 
 
 
 
 
 
61 
13.0 SUPPLEMENTARY TABLES 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
62 
 
 

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1.0 TRIAL SUMMARY 
Abbreviated Title 
Pembrolizumab effects on NK cell exhaustion in melanoma 
Trial Phase 
II 
Clinical Indication 
Unresectable stage III and stage IV 
Trial Type 
Treatment 
Type of control 
No control arm 
Route of administration 
IV 
Trial Blinding 
Not blinded 
Treatment Groups  
Pembrolizumab 200 mg every 3 weeks 
Number of trial subjects 
125, 100 melanoma patients and 25 healthy donors 
Estimated enrollment period 
3 yr 
Estimated duration of trial 
5 yr 
Duration of Participation 
5 yr (includes OS follow up) 
 
 
 
2.0 TRIAL DESIGN 
2.1 
Trial Design 
100 patients with unresectable stage III or stage IV melanoma will receive intravenous 
treatment with pembrolizumab at a dose of 200 mg every 3 weeks. This study consists of 
several treatment cycles, where each treatment cycle is defined as 21 days (3 weeks). 
Therefore treatment cycles 1,2,3,4, 5, 6,7,8, 9 are defined as starting respectively at week 1, 
4,7,10, 13, 16, 19, 22, 25. Treatment will be continued every 3 weeks unless there is a reason 
to discontinue. Once the drug is discontinued there will be follow up visits. There will be one 
visit at the time the drug is discontinued (Discontinuation visit), one Safety Follow up Visit, 
and subsequent Follow up visits every 12 weeks until progression or if patient withdraws 
from the study or up to 3 years after enrollment. Patient will be called about every 12 weeks 
to monitor their survival. Blood samples for cell profiling , plasma measurements and 
transcriptome analysis will be collected at baseline (2 values; visit 2 and prior to cycle 1) and 
prior every cycle, to assess for: (a) NK cell exhaustion phenotype; (b) plasma levels of 
MICA, HMGB1 and CEACAM-1; (c) ALC; (d) MDSC frequency and (e) frequency of 
ICOS+CD4+ and EOMES+ CD8+ T cells.  
Clinical responses will be assessed according to Immune-Related Response Criteria. Patients 
who fail to receive the first 4 cycles of treatment due to toxicity of treatment will be replaced. 
All patients will be followed until progression or up to 3 years of follow up. 
Changes in the NK cell exhaustion phenotype will be correlated with best overall response as 
a primary endpoint and with 24 week progression free survival and 1-year overall survival as 
secondary endpoints. 

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Analysis of NK cells will comprise assessment of phenotypic and functional parameters. 
Phenotypic characterization includes expression of exhaustion markers Tim-3, CEACAM-1, 
IL-2R subunits, activating and inhibitory NK cell receptors (NKR). Functional analysis 
includes cytokine production, cytotoxicity and proliferation. These studies will be 
supplemented by CyTOF analysis to validate pathways associated with the exhaustion state.  
Biomarkers associated with the NK cell exhausted state will be monitored in plasma by 
ELISA (MICA, HMGB1 and CEACAM-1).  
We expect to see a clinical response rate of 27% when measured by immune related response 
criteria 1. An inverse correlation of clinical response with pre-treatment plasma CEACAM-1, 
HMGB-1 and MICA levels is projected. We anticipate that responding patients will display 
previously established biomarkers of response (higher ALC, ICOS+ CD4+ T cells, EOMES+ 
CD8+ T cells, and lower levels of MDSC).  
25 healthy donors will be recruited into the study to donate blood leukocytes and plasma in 
order to provide baseline “normal” ranges and parameters of NK cell phenotype and 
function, biomarker distribution, ALC levels, MDSC levels and frequency of ICOS+CD4+ 
and EOMES+ CD8+ T cells.   
 
2.2 
Trial Diagram 
 
 
 
 
 
 
• Research blood samples for cell profiling and plasma measurements will be obtained 
at baseline (2 values; visit 2 and prior to cycle 1), week 4 (prior to cycle 2), week 7 
(prior to cycle 3), week 10 (prior to cycle 4), week 13 (prior to cycle 5), week 16 
(prior to cycle 6), week 19 (prior to cycle 7), week 22 (prior to cycle 8), week 25 
(prior to cycle 9), week 34 (prior to infusion 10), week 52, week 78, week 104, and at 
end of treatment and 30 days post discontinuation. Imaging studies to assess efficacy 
of treatment will be performed during week 3 of every 4th treatment cycle while 
receiving pembrolizumab infusion. 
 
Unresectable 
stage III  
or  
StageIV 
melanoma 
Screening 
Pembrolizumab  
200 mg q 3 weeks 

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3.0 OBJECTIVES & HYPOTHESES 
3.1 
Primary Objective & Hypothesis 
(1) Objective: To correlate changes in the NK cell exhaustion phenotype with the 
clinical efficacy (best overall response) of pembrolizumab treatment in patients with 
Unresectable stage III or IV melanoma using Immune-Related Response Criteria.  
Hypothesis: Pharmacodynamic reversal of NK cell exhaustion is associated with                     
clinical responsiveness to pembrolizumab induced PD-1 checkpoint blockade. 
 
3.2 
Secondary Objectives & Hypotheses 
(1) 
Objective: To identify biomarkers associated with a reversal of NK cell      
exhaustion in vivo following treatment with pembrolizumab.  
Hypothesis:  Reversal of NK cell exhaustion is correlated with biomarkers such as 
HMGB-1, CEACAM-1 and MICA.  
(2)  
Objective: To determine how treatment with pembrolizumab modulates NK cell         
exhaustion in patients with stage IV melanoma.  
Hypothesis: The metastatic melanoma microenvironment induces the development    
of exhausted NK cells. 
(3)    Objective: To identify Tim-3 associated and non-associated molecular or protein   
targets that potentiate this phenotype.  
Hypothesis: The development of exhausted NK cells in the microenvironment of a    
melanoma metastasis is potentiated by protein targets, including Tim-3 associated 
and non-associated targets.  
(4) 
Objective: To correlate changes in the NK cell exhaustion phenotype with one   
year survival following pembrolizumab treatment in patients with Unresectable 
stage III or IV melanoma using Immune-Related Response Criteria. 
Hypothesis: Pharmacodynamic reversal of NK cell exhaustion is associated with                   
improved survival following pembrolizumab induced PD-1 checkpoint blockade. 
(5) 
Objective: To correlate changes in the NK cell exhaustion phenotype with 6 
month progression free survival (PFS) in pembrolizumab treated patients with 
Unresectable stage III or IV melanoma using Immune-Related Response Criteria. 

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   Hypothesis:  Pharmacodynamic reversal of NK cell exhaustion is associated with                  
improved PFS in response to pembrolizumab induced PD-1 checkpoint blockade. 
(6) 
Objective: To corroborate the presence of exhausted NK cells within the tumor and 
its reversal after treatment. 
   Hypothesis:  NK cells in the tumor microenvironment will be exhausted but 
function and frequency can be restored with pembrolizumab treatment.  
As a control for these exploratory studies, NK cells and plasma will be obtained 
from peripheral blood of 25 healthy donors (HD). 
 
4.0 BACKGROUND & RATIONALE 
4.1 
Background 
4.1.1 Melanoma 
It is estimated that in 2014 approximately 76,100 people in the United States will be 
diagnosed with melanoma and 9710 will die because of the malignancy. The median life 
expectancy for stage IV melanoma (distant metastases) is under one year. Prior to 2011 only 
two agents, dacarbazine and high dose interleukin-2 (HD-IL2), were Food and Drug 
Administration (FDA) approved for the management of stage IV melanoma. The rate of 
response to dacarbazine chemotherapy is less than 20% and the responses are largely partial 
and treatment does not confer an overall survival benefit. HD-IL2 is a cytokine based 
treatment requiring in-patient administration and confers a 16% response rate with a 5% 
durable complete response rate.  
Approximately 50% of melanomas select for an activating mutation at position 600 of BRAF 
(V600) leading to constitutive activation of the Mitogen Activated Protein Kinase signaling 
pathway (MAPK pathway). Targeted inhibition of BRAF or the combination of BRAF plus 
MEK confers survival benefits in patients with melanoma expressing a V600 BRAF 
mutation. The rate of response to dual BRAF and MEK inhibition using dabrafenib and 
trametinib is approximately 67% with median duration approximately 10 months.  
In 2011, ipilimumab, which is a monoclonal antibody which targets and blocks CTLA-4, was 
FDA approved for the management of stage IV melanoma. CTLA4 is a checkpoint molecule 
that down regulates the activation of T-cells. In a phase III trial, treatment of stage IV 
melanoma patients with ipilimumab resulted in a statistically significant overall survival 
benefit relative to treatment with a peptide vaccine. The median overall survival increased 
from 6.4 months to 10.1 months and 2-year survival increased from 13.7% to 23.5% of 
patients.  

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In 2014, two immunotherapies targeting PD-1, nivolumab and pembrolizumab, were FDA 
approved for the treatment of stage IV melanoma. Additional background information is 
provided below.  
In 2015, pembrolizumab was approved as initial treatment of patients with unresectable or 
metastatic melanoma. 
4.1.2 NK Cells 
NK cells are key participants in the initial immune response to tumorigenesis, equipped to 
respond rapidly through induction of cytokines and deployment of cytotoxic activity2. A 
dynamic balance of positive and negative signals regulates NK cell target cell recognition 
and lysis, through engagement of a variety of activation and inhibitory receptors (NK cell 
receptors; NKR). NK cells detect a state of ‘missing-self’ by MHC class I-specific inhibitory 
receptors and they recognize cell stress-induced self-ligands on cancer cells via their 
activating receptors. NK cells contribute to containing tumor growth as NK cell infiltration in 
tumor tissue correlates with better prognosis, whereas reduced activity of peripheral blood 
NK cells is associated with an increased risk of cancer3. We have now obtained clear 
evidence that NK cells progressively undergo a process of “exhaustion” that coincides 
with disease progression from early to late stage melanoma. Freshly purified NK cells 
from 113 patients with untreated melanoma (melanoma donors; stages I, II and III/IV) 
were analyzed for this study. Similar to exhausted T cells, melanoma associated NK cells 
are characterized by a failure to proliferate, to produce interferon gamma (IFNγ) or to kill 
target cells. They (i) down regulate activation receptors (CD16, NKG2D, NKp46, and 
DNAM-1), IL-2R subunits and NK cell regulatory transcription factors (T-bet, Eomes), (ii) 
upregulate inhibitory receptors (KIR3DL1, KIR2DL3) and (iii) express high levels of the 
checkpoint molecule Tim-3, a phenotype that is consistent with “NK cell exhaustion”. 
Significantly, blockade of Tim-3 reverses this phenotype and state of exhaustion in 
vitro, restoring 30-65% of the NK cell dysfunction4. Most strikingly however, we find that 
NK cell function is spontaneously restored in patients who have a clinical response to 
ipilimumab treatment despite the fact that these cells express little or no CTLA-4 (or PD-1 or 
PDL-1), possibly through reversal of tumor-associated systemic immune suppression. 
Characterizing NK cell dysfunction in melanoma, will be critical to understanding the 
modulation of NK cell biology in the tumor microenvironment (TME), and, ultimately, 
developing approaches that restore both innate and adaptive immunity, in vivo.   
PRELIMINARY DATA: 
Defining the spectrum of NK cell exhaustion in melanoma and identification of 
molecular or protein targets that potentiate NK cell exhaustion. 
NK cell exhaustion in melanoma:  Blood NK cells from melanoma patients develop a 
phenotypic and functional profile consistent with progressive exhaustion from stage I to stage 
IV (Figure 1).  This is characterized by reductions in:  

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(i) cytotoxicity (expression of LAMP-1/CD107a in response to co-culture with K562 cells), 
(ii) IFNγ production in response to IL-12, and  
(iii) proliferation in response to IL-2, consistent with the concept that advanced disease is 
progressively associated with systemic immune suppression (Figure 1).  
The exhaustion profile is global as it is distributed through both CD56hi and CD16 hi NK 
cell subsets (data not shown) 4.  
 
 
NK cell exhaustion and Tim-3: In an analysis of stage III/IV melanoma patients, NK cell 
exhaustion was found to be directly associated with up regulation of checkpoint molecules 
(Tim-3), reduced NK cell cytotoxic activity, IFNγ production, and proliferative capacity, and 
lower levels of T-bet (Supplementary Table 1). Elevated Tim-3 was also significantly 
associated with expression of the inhibitory receptor KIRB1 and poor prognostic factors, 
such as, tumor thickness >1mm, mitotic rate >/= 1/mm2, ulceration and metastases 
(Supplementary Table 2).  
Strikingly, Tim-3 blockade substantially reversed exhaustion, improving NK cell 
cytotoxicity, IFNγ production and IL-2 dependent proliferation (Figure 2). Reversal was 
associated with up regulation of the activating CD16 receptor and α and γ chains of IL-2R4. 
These data denote a major role for Tim-3 in modulating NK cell function in melanoma. 
 
 
Figure 1 a-c: NK cells progressively develop a phenotypic and functional profile consistent with 
exhaustion. Represented by a) cytotoxicity b) IFNγ production c) proliferation.  Patient numbers: Stage I 
(n=56) Stage II (n=21) Stage III/IV (n=23). 
Figure 2: Tim-3 blockade partially reverses NK cell exhaustion. Anti-Tim-3 (20µg/ml) reverses 
blockade of (a) cytotoxicity, (b) cytokine production and (c) proliferation of melanoma NK cells. 

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Tim-3 ligation inhibits NK cell function: Tim-3 ligands include galectin-95, HMGB-1 (high 
mobility group box-1, released by necrotic tumor cells)6, phosphatidylserine and CEACAM-
1 (carcinoembryonic antigen cell adhesion molecule 1), which functions in cis and trans 
configurations as a heterophilic ligand to promote the tolerance-inducing functions of TIM-
37. We have confirmed that CEACAM-1 is expressed on normal NK cells and its expression 
level increases on IL-2 and/or IL-15 activated CD56hi NK cells as previously described8 
(Figure 3).  
 
 
 
 
 
 
We found NK cell mediated cytotoxicity was significantly reduced in Gal-9+ but not Gal9- 
Gmel melanoma cells, supporting a role for Tim-3-galectin 9 engagement in the potentiation 
of NK cell exhaustion.4 To address the role of other NKR ligands, we evaluated plasma 
levels of HMGB1 and MICA, a known NKG2D ligand produced by melanoma cells and 
associated with dysfunctional NK cells in melanoma9 (Figure 4). Levels of both proteins rose 
progressively with advancing stage of disease, and an independent cohort of patients (n=204) 
validated the association of higher levels of MICA (>500 pg/ml) with worse prognosis (data 
not shown). Altogether these data warrant a closer examination of the role of Tim-3 and 
NKR ligands in mediating exhaustion and as possible prognostic biomarkers. 
Figure 3: Detection of Tim-3 and CEACAM-1 in human NK cells and 721.221 cell line by 
Flow Cytometry. NK cells were freshly isolated from a healthy donor’s blood (a) then 
stimulated O/N with (b) rhIL-2 (1000U/ml) or (c) rhIL-15 (100ng/ml).  (d) CEACAM-1 
expression in the 721.221 cell line that stably over-expresses CEACAM-1 is shown as a positive 
control. 

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Biomarkers associated with reversal of NK cell exhaustion through check-point 
blockade. 
Preliminary studies indicate that ipilimumab, an anti-CTLA-4 monoclonal antibody, can 
reverse blood NK cell exhaustion in melanoma patients.  Blood NK cells from stage IV 
donors recovered their natural response to IL-2 stimulation (500U/ml) over 7 days after 3 
cycles of ipilimumab (Figure 5). Recovered responses included up regulation of IL-2 R α and 
γ chains and activating NKR (NKG2D and NKp46), cytotoxicity (Lamp-1 expression) and 
IFNγ production. In contrast, NK cells from patients who failed to respond to ipilimumab did 
not restore IL-2R α chain expression in response to IL-2 (not shown).  As ipilimumab 
resistant patients can respond to subsequent anti-PD-1 therapy 1,10, we propose that clinical 
responders to anti-PD-1 treatment will have effectively reversed NK cell exhaustion.  
 
Figure 4: MICA and HMGB1 levels in plasma from melanoma patients.  Levels were 
measured by ELISA. The numbers of patients evaluated in each cohort are: (a) Stage I (n=72), 
Stage II (n=24), Stage III/IV (n=15); (b) Stage I (n=36), Stage II (n=11), Stage III/IV (n=14); (c) 
an independent validation cohort shows that MICA >500 pg/ml predicts OS.  
Figure 5 (a-i). NK-cell exhaustion can be reversed following treatment with 3 cycles of ipilimumab. NK 
cells were cultured with IL-2 and phenotype and function monitored over time. Black solid line (HD=healthy 
donor); Black dashed line (MD= Melanoma donor); Red solid line (MD treated=Melanoma donor). (j) NK-
cell exhaustion is rapidly reversed with ipilimumab. NK cells from patients pre/post 1, 2, 3 or 4 cycles of 
Ipilimumab at 3 week intervals were cultured with IL-2. Levels of IL-2Rα chain were monitored over 6d. 

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Our studies indicate that MD (melanoma donor) NK cells exhibit a progressively exhausted 
phenotype characterized by up-regulation of inhibitory receptors (TIM-3, KIRB1 and 
KIRNKAT2), down-regulation of activating receptors (NKG2D and NKp46), IL-2R subunits 
and transcription factors (T-bet and Eomes), and loss of IFNγ production, proliferation and 
cytotoxicity. Tim-3 expression, which is critical to conferring this exhausted NK cell 
phenotype, is associated with poor prognostic markers of overall survival (OS). Systemic 
elevation of NKR ligands such as MICA (NKG2D ligand) and HMGB1 (Tim-3 ligand) may 
portend worse outcome by potentiating NK cell exhaustion. Checkpoint blockade can reverse 
this phenotype, even in the absence of CTLA-4 or PD-1 on NK cells, warranting further 
investigation of parameters that may predict this outcome.  
The primary objective of this proposal, therefore, is to correlate changes in the NK cell 
exhaustion phenotype with the clinical efficacy (best overall response) of pembrolizumab 
treatment in patients with Unresectable stage III or IV melanoma using Immune-Related 
Response Criteria. Secondary objectives include identifying biomarkers associated with a 
reversal of NK cell exhaustion in vivo following treatment, identifying the associated 
proteins that potentiate this phenotype, and evaluating tumor associated NK cells, if feasible.  
A cohort of 25 healthy donors will be included to determine the normal ranges for the 
biomarkers and proteins associated with the NK exhaustion phenotype. 
 
4.1.3 Pharmaceutical and Therapeutic Background (pembrolizumab) 
The importance of intact immune surveillance in controlling outgrowth of neoplastic 
transformation has been known for decades.  Accumulating evidence shows a correlation 
between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in 
various malignancies.  In particular, the presence of CD8+ T-cells and the ratio of CD8+ 
effector T-cells / FoxP3+ regulatory T-cells seems to correlate with improved prognosis and 
long-term survival in many solid tumors. 
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress 
immune control.  The normal function of PD-1, expressed on the cell surface of activated T-
cells under healthy conditions, is to down-modulate unwanted or excessive immune 
responses, including autoimmune reactions.  PD-1 (encoded by the gene Pdcd1), an Ig 
superfamily member related to CD28 and CTLA-4, has been shown to negatively regulate 
antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2).  The 
structure of murine PD-1 has been resolved.  PD-1 and its family members are type I 
transmembrane glycoproteins containing an Ig Variable-type (V-type) domain responsible 
for ligand binding and a cytoplasmic tail which is responsible for the binding of signaling 
molecules.  The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an 
immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor tyrosine-
based switch motif (ITSM).  Following T-cell stimulation, PD-1 recruits the tyrosine 

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phosphatases SHP-1 and SHP-2 to the ITSM motif within its cytoplasmic tail, leading to the 
dephosphorylation of effector molecules such as CD3ζ, PKCθ and ZAP70, which are 
involved in the CD3 T-cell signaling cascade.  The mechanism by which PD-1 down 
modulates T-cell responses is similar to, but distinct from, that of CTLA-4 as both molecules 
regulate an overlapping set of signaling proteins.  PD-1 was shown to be expressed on 
activated lymphocytes; including peripheral CD4+ and CD8+ T-cells, B-cells, T regs and 
Natural Killer cells.  Expression has also been shown during thymic development on CD4-
CD8- (double negative) T-cells as well as subsets of macrophages and dendritic cells.  The 
ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in a 
variety of cell types, including non-hematopoietic tissues as well as various tumors.  Both 
ligands are type I transmembrane receptors containing both IgV- and IgC-like domains in the 
extracellular region and contain short cytoplasmic regions with no known signaling motifs.  
Binding of either PD-1 ligand to PD-1 inhibits T-cell activation normally triggered through 
the T-cell receptor.  PD-L1 is expressed at low levels on various non-hematopoietic tissues, 
most notably on vascular endothelium, whereas PD-L2 protein is only detectably expressed 
on antigen-presenting cells found in lymphoid tissue or chronic inflammatory environments.  
PD-L2 is thought to control immune T-cell activation in lymphoid organs, whereas PD-L1 
serves to dampen unwarranted T-cell function in peripheral tissues.  Although healthy organs 
express little (if any) PD-L1, a variety of cancers were demonstrated to express abundant 
levels of this T-cell inhibitor.  PD-1 has been suggested to regulate tumor-specific T-cell 
expansion in subjects with melanoma (MEL).  This suggests that the PD-1/PD-L1 pathway 
plays a critical role in tumor immune evasion and should be considered as an attractive target 
for therapeutic intervention. 
Pembrolizumab is a potent and highly selective humanized monoclonal antibody (mAb) of 
the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its 
ligands, PD-L1 and PD-L2. On December 18, 2015, the U. S. Food and Drug Administration 
(FDA) expanded the label to include the approval of pembrolizumab for the treatment of 
patients with unresectable or metastatic melanoma. This expansion now includes the initial 
treatment of patients with unresectable or metastatic melanoma with pembrolizumab.  
Refer to the Investigator’s Brochure (IB)/approved labeling for detailed background 
information on MK-3475 (pembrolizumab). 
 
4.1.4 Preclinical and Clinical Trial Data 
Refer to the Investigator’s Brochure for Preclinical and Clinical data. 
4.2 
Rationale 
Antibodies targeting the checkpoint molecules CTLA-4 and PD-1 have dramatically 
improved the treatment of metastatic melanoma, although there are subsets of patients who 

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fail to respond. Identifying parameters that predict a durable response are urgently needed. 
We observed rapid reversal of NK cell exhaustion in patients with a clinical response 
following treatment with anti-CTLA-4, even though NK cells do not express this molecule. 
However, patients who failed to respond clinically also failed to reverse this exhaustion 
profile.  We propose that early reversal of function may predict clinical response to anti-PD-1 
therapy in patients who have either failed ipilimumab or other therapies, or who are ineligible 
for these therapies. Progressive melanoma is characterized by elevated levels of plasma 
CEACAM-1, MICA and HMGB1, all known ligands for various NKR, and which have been 
associated with worse OS. NKR ligand levels, therefore, may function as pretreatment 
biomarkers for checkpoint blockade inhibition.  
4.2.1 Rationale for the Trial and Selected Subject Population 
Melanoma is an immune-modulated malignancy and immune checkpoint modulators which 
inhibit PD-1 function  (pembrolizumab, nivolumab) have demonstrated clinical efficacy as 
treatment for patients with stage IV melanoma. Pembrolizumab across a range of doses in 
phase I investigation has demonstrated clinical efficacy with RR approximately 27%. By 
better understanding how NK cell function and exhaustion interplays with PD1 function and 
activity, potentially more efficacious combination therapies can be developed. The 
pharmacodynamic studies to be performed as part of this trial will provide such information.  
4.2.2 Rationale for Dose Selection/Regimen/Modification  
An open-label Phase I trial (Protocol 001, PN001) is being conducted to evaluate the safety 
and clinical activity of single agent MK-3475.  The dose escalation portion of this trial 
evaluated three dose levels, 1 mg/kg, 3 mg/kg, and 10 mg/kg, administered every 2 weeks 
(Q2W) in subjects with advanced solid tumors.  All three dose levels were well tolerated and 
no dose-limiting toxicities were observed.  This first in human study of MK-3475 showed 
evidence of target engagement and objective evidence of tumor size reduction at all dose 
levels (1 mg/kg, 3 mg/kg and 10 mg/kg Q2W).  No MTD has been identified to date.  10.0 
mg/kg Q2W, the highest dose tested in PN001, was chosen as the dose and schedule utilized 
in Cohorts A, B, C and D of that protocol to test for initial tumor activity.  Recent data from 
other clinical studies within the MK-3475 program has shown that a lower dose of MK-3475 
and a less frequent schedule may be sufficient for target engagement and clinical activity. 
 
PK data analysis of MK-3475 administered both Q2W and Q3W showed slow systemic 
clearance, limited volume of distribution, and a long half-life (refer to IB).  
Pharmacodynamic data (IL-2 release assay) suggested that peripheral target engagement is 
durable (>21 days).  This early PK and pharmacodynamic data provided the scientific 
rationale for testing a Q2W and Q3W dosing schedule.   
 
A population pharmacokinetic analysis has been performed using serum concentration time 
data from 476 patients. Within the resulting population PK model, clearance and volume 
parameters of MK-3475 were found to be dependent on body weight. The relationship 

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between clearance and body weight, with an allometric exponent of 0.59, is within the range 
observed for other antibodies and would support both body weight normalized dosing or a 
fixed dose across all body weights.  MK-3475 has been found to have a wide therapeutic 
range based on the melanoma indication.  The differences in exposure for a 200 mg fixed 
dose regimen relative to a 2 mg/kg Q3W body weight based regimen are anticipated to 
remain well within the established exposure margins of 0.5 – 5.0 for MK-3475 in the 
melanoma indication. The exposure margins are based on the notion of similar efficacy and 
safety in melanoma at 10 mg/kg Q3W vs. the dose regimen of 2 mg/kg Q3W (i.e. 5-fold 
higher dose and exposure). The population PK evaluation revealed that there was no 
significant impact of tumor burden on exposure. In addition, exposure was similar between 
the NSCLC and melanoma indications. Therefore, there are no anticipated changes in 
exposure between different indication settings. 
 
The rationale for further exploration of 2 mg/kg and comparable doses of pembrolizumab in 
solid tumors was based on: 1) similar efficacy and safety of pembrolizumab when dosed at 
either 2 mg/kg or 10 mg/kg Q3W in melanoma patients, 2) the flat exposure-response 
relationships of pembrolizumab for both efficacy and safety in the dose ranges of 2 mg/kg 
Q3W to 10 mg/kg Q3W, 3) the lack of effect of tumor burden or indication on distribution 
behavior of pembrolizumab (as assessed by the population PK model) and 4) the assumption 
that the dynamics of pembrolizumab target engagement will not vary meaningfully with 
tumor type. 
The choice of 200 mg Q3W as an appropriate dose for the switch to fixed dosing is based on 
simulations performed using the population PK model of pembrolizumab showing that a 
fixed dose of 200 mg every 3 weeks will provide exposures that 1) are optimally consistent 
with those obtained with the 2 mg/kg dose every 3 weeks, 2) will maintain individual patient 
exposures in the exposure range established in melanoma as associated with maximal 
efficacy response and 3) will maintain individual patients exposure in the exposure range 
established in melanoma that are well tolerated and safe.  
A fixed dose regimen will simplify the dosing regimen to be more convenient for physicians 
and to reduce the potential for dosing errors.  A fixed dosing scheme will also reduce 
complexity in the logistical chain at treatment facilities and reduce wastage. 
 
 
5.0 METHODOLOGY 
5.1 
Entry Criteria 
5.1.1 Melanoma Subject Inclusion Criteria 
     In order to be eligible for participation in this trial, the subject must: 

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1. Be willing and able to provide written informed consent/assent for the trial. 
2. Have Unresectable stage III or stage IV melanoma 
3. Be ≥ 18 years of age on day of signing informed consent. 
4. Have measurable disease based on RECIST 1.1 and be able to be followed over time 
by Immune related response criteria (irRC) for treatment decisions. 
5. Have a performance status of 0, 1, or 2 on the ECOG Performance Scale. 
6. Demonstrate adequate organ function as defined in Table 1, all screening labs should 
be performed within 10 days of treatment initiation. 
 
Table 1 - Adequate Organ Function Laboratory Values 
System 
Laboratory Value 
Hematological 
 
Absolute neutrophil count (ANC) 
≥1,500 /mcL 
Platelets 
≥100,000 / mcL 
Hemoglobin 
≥9 g/dL or ≥5.6 mmol/L without transfusion or EPO dependency 
(within 7 days of assessment) 
Renal 
 
Serum creatinine OR 
Measured or calculateda creatinine 
clearance 
(GFR can also be used in place of 
creatinine or CrCl) 
≤1.5 X upper limit of normal (ULN) OR 
 
≥60 mL/min for subject with creatinine levels > 1.5 X 
institutional ULN 
Hepatic 
 
Serum total bilirubin 
≤ 1.5 X ULN OR 
 
Direct bilirubin ≤ ULN for subjects with total bilirubin levels > 
1.5 ULN 
AST (SGOT) and ALT (SGPT) 
≤ 2.5 X ULN  OR 
≤ 5 X ULN for subjects with liver metastases 
Albumin 
>2.5 mg/dL 
Coagulation 
International Normalized Ratio (INR) or 
Prothrombin Time (PT) 
 
Activated Partial Thromboplastin Time 
(aPTT) 
≤1.5 X ULN unless subject is receiving anticoagulant therapy 
as long as PT or PTT is within therapeutic range of intended use 
of anticoagulants 
≤1.5 X ULN unless subject is receiving anticoagulant therapy 
as long as PT or PTT is within therapeutic range of intended use 
of anticoagulants 
aCreatinine clearance should be calculated per institutional standard. 
 
7. Female subjects of childbearing potential should have a negative urine or serum 
pregnancy within 72 hours prior to receiving the first dose of study medication.  If the 
urine test is positive or cannot be confirmed as negative, a serum pregnancy test will 
be required.   

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8. Female subjects of childbearing potential should be willing to use 2 methods of birth 
control or be surgically sterile, or abstain from heterosexual activity for the course of 
the study through 120 days after the last dose of study medication (Reference Section 
5.6.2).  Subjects of childbearing potential are those who have not been surgically 
sterilized or have not been free from menses for > 1 year.  
9. Male subjects should agree to use an adequate method of contraception starting with 
the first dose of study therapy through 120 days after the last dose of study therapy. 
5.1.2 Melanoma Subject Exclusion Criteria 
The subject must be excluded from participating in the trial if the subject: 
1. Is currently participating and receiving study therapy or has participated in a study of 
an investigational agent and received study therapy or used an investigational device 
within 4 weeks prior to the first dose of treatment. 
2. Has a diagnosis of immunodeficiency or is receiving systemic steroid therapy or any 
other form of systemic immunosuppressive therapy within 7 days prior to the first 
dose of trial treatment. Topical, inhaled, ocular, and intra-articular steroids are not 
exclusionary. 
3. Has a known history of active TB   (Bacillus Tuberculosis). 
 
4. Hypersensitivity to pembrolizumab or any of its excipients. 
5. Has had a prior anti-cancer monoclonal antibody (mAb) treatment within 4 weeks 
prior to study Day 1 or who has not recovered (i.e., ≤ Grade 1 or at baseline) from 
adverse events due to agents administered more than 4 weeks earlier. 
6. Has had prior chemotherapy, targeted small molecule therapy, or radiation therapy 
within 2 weeks prior to study Day 1 or who has not recovered (i.e., ≤ Grade 1 or at 
baseline) from adverse events due to a previously administered agent. 
- 
Note:  Subjects with ≤ Grade 2 neuropathy are an exception to this criterion 
and may qualify for the study. 
- 
Note:  If subject received major surgery, they must have recovered adequately 
from the toxicity and/or complications from the intervention prior to starting 
therapy.   
7. Has a known additional malignancy that is progressing or requires active treatment.  
Exceptions include basal cell carcinoma of the skin or squamous cell carcinoma of 
the skin that has undergone potentially curative therapy or in situ cervical cancer. 

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8. Has known active central nervous system (CNS) metastases and/or carcinomatous 
meningitis. Subjects with previously treated brain metastases may participate 
provided they are stable (without evidence of progression by imaging for at least four 
weeks prior to the first dose of trial treatment and any neurologic symptoms have 
returned to baseline), have no evidence of new or enlarging brain metastases, and are 
not using steroids for at least 7 days prior to trial treatment.  This exception does not 
include carcinomatous meningitis, which is excluded regardless of clinical stability.   
9. Has active autoimmune disease that has required systemic treatment in the past 2 
years 
(i.e. 
with 
use 
of 
disease 
modifying 
agents, 
corticosteroids 
or 
immunosuppressive drugs). Replacement therapy (eg. thyroxine, insulin, or 
physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, 
etc.) is not considered a form of systemic treatment.  
10. Has a history of (non-infectious) pneumonitis that required steroids or current 
pneumonitis. 
11. Has an active infection requiring systemic therapy. 
12. Has a history or current evidence of any condition, therapy, or laboratory abnormality 
that might confound the results of the trial, interfere with the subject’s participation 
for the full duration of the trial, or is not in the best interest of the subject to 
participate, in the opinion of the treating investigator.   
13. Has known psychiatric or substance abuse disorders that would interfere with 
cooperation with the requirements of the trial. 
14. If 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. 
15. Has received prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent. 
16. Has a known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies). 
17. Has known active Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., HCV RNA 
[qualitative] is detected). 
18. 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. 

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5.1.3 Healthy subject inclusion and exclusion criteria.  
25 healthy volunteers will serve as the “baseline” healthy population for comparison 
purposes to assess NK cell function and protein biomarkers in the blood. They will not 
received trial treatment. 
In order to be eligible for participation in this trial, the subject must: 
1. Be willing and able to provide written informed consent/assent for the trial. 
2. Be ≥ 18 years of age on day of signing informed consent. 
3. Be free of chronic illness, without known heart, lung, kidney, bleeding disorders, 
infectious disease (HIV, HBV or HCV infection).  
4. Not taking regularly prescribed medication such as steroids, hormone therapy or 
immunosuppressive agents. 
5. Not pregnant or breastfeeding. 
6. Have adequate organ function   (see Table 1). 
5.2 
Trial Treatments 
The treatment to be used in this trial is outlined below in Table 2Table 2 
Table 2 - Trial Treatment 
Drug 
Dose/Potency 
Dose 
Frequency 
Route of 
Administration 
Regimen/Treatment 
Period 
Use 
Pembrolizumab 200 mg  
Q3W 
IV infusion 
Day 1 of each 3 week 
cycle 
Experimental 
 
All enrolled melanoma subjects will receive treatment with pembrolizumab at the dose of 
200mg intravenous every 3 weeks.  
5.2.1 Dose Selection/Modification 
5.2.1.1 Dose Selection 
The rationale for selection of doses to be used in this trial is provided in Section 4.0 – 
Background and Rationale.   
Details on preparation and administration of pembrolizumab (MK-3475) are provided in the 
Pharmacy Manual.   

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5.2.1.2 Dose Modification  
Adverse events (both non-serious and serious) associated with pembrolizumab exposure may 
represent an immunologic etiology.  These adverse events may occur shortly after the first 
dose or several months after the last dose of treatment. Pembrolizumab must be withheld for 
drug-related toxicities and severe or life-threatening AEs as per Table 3 below. See Section 
5.5.1 and the Investigator Brochure for MK-3475 for supportive care guidelines, including 
use of corticosteroids. 
Table 3 - Dose Modification Guidelines for Drug-Related Adverse Events 
Toxicity 
Hold 
Treatment 
For Grade 
Timing for Restarting Treatment 
Discontinue Subject 
Diarrhea/Colitis 
2-3 
 
Toxicity resolves to Grade 0-1. 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks. 
4 
Permanently discontinue 
Permanently discontinue 
AST, ALT, or 
Increased Bilirubin 
2 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose. 
3-4 
Permanently discontinue 
(see exception below)1 
Permanently discontinue 
Type 1 diabetes 
mellitus (if new 
onset) or 
Hyperglycemia 
T1DM or  
3-4 
Hold pembrolizumab for new onset Type 
1 
diabetes 
mellitus 
or 
Grade 
3-4 
hyperglycemia associated with evidence of 
beta cell failure.  
 
Resume pembrolizumab when patients are clinically and 
metabolically stable. 
Hypophysitis 
2-3 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks. 
4 
Permanently discontinue 
Permanently discontinue 
Hyperthyroidism  
3 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks. 
4 
Permanently discontinue 
Permanently discontinue 
Hypothyroidism 
2-4 
Therapy with pembrolizumab can be 
continued while treatment for the thyroid 
disorder is instituted 
Therapy with pembrolizumab can be continued while treatment for 
the thyroid disorder is instituted. 
Infusion Reaction 
3-4 
Permanently discontinue 
Permanently discontinue 
Pneumonitis 
2 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks. 
3-4 
Permanently discontinue 
Permanently discontinue 
Renal Failure or 
Nephritis 
2 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks. 
3-4 
Permanently discontinue 
Permanently discontinue 
All Other Drug-
Related Toxicity2 
3 or Severe 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last dose or inability 
to reduce corticosteroid to 10 mg or less of prednisone or equivalent 
per day within 12 weeks. 
4 
Permanently discontinue 
Permanently discontinue 
Note: Permanently discontinue for any severe or Grade 3 drug-related AE that recurs or any life-threatening event. 
1 For patients with liver metastasis who begin treatment with Grade 2 AST or ALT, if AST or ALT increases by greater than or equal to 50% relative to baseline 
and lasts for at least 1 week then patients should be discontinued. 

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Toxicity 
Hold 
Treatment 
For Grade 
Timing for Restarting Treatment 
Discontinue Subject 
2 Patients with intolerable or persistent Grade 2 drug-related AE may hold study medication at physician discretion.  Permanently discontinue study drug for 
persistent Grade 2 adverse reactions for which treatment with study drug has been held, that do not recover to Grade 0-1 within 12 weeks of the last dose.   
 
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, patient 
vacation, and/or holidays). Subjects should be placed back on study therapy within 3 weeks 
of the scheduled interruption, unless otherwise discussed with the Sponsor. The reason for 
interruption should be documented in the patient's study record. 
5.2.2 Timing of Dose Administration 
Trial treatment should be administered on Day 1 of each cycle after all 
procedures/assessments have been completed as detailed on the Trial Flow Chart (Section 
6.0).  Trial treatment may be administered up to 3 days before or after the scheduled Day 1 of 
each cycle due to administrative reasons. 
All trial treatments will be administered on an outpatient basis. 
Pembrolizumab 200 mg will be administered as a 30 minute IV infusion every 3 weeks.  
Sites should make every effort to target infusion timing to be as close to 30 minutes as 
possible.  However, given the variability of infusion pumps from site to site, a window of -5 
minutes and +10 minutes is permitted (i.e., infusion time is 30 minutes: -5 min/+10 min). 
The Pharmacy Manual contains specific instructions for the preparation of the 
pembrolizumab infusion fluid and administration of infusion solution. 
5.2.3 Trial Blinding/Masking 
This is a single agent, open-label trial; therefore, the Sponsor, investigator and subject will 
know the treatment administered. 
5.3 
Randomization or Treatment Allocation 
There is no randomization. All enrolled melanoma subjects will receive treatment with 
pembrolizumab at the dose of 200 mg intravenous every 3 weeks.  
5.4 
Concomitant Medications/Vaccinations (allowed & prohibited) 
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 

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with the Merck Clinical team.  The final decision on any supportive therapy or vaccination 
rests with the investigator and/or the subject's primary physician.  
5.4.1 Acceptable Concomitant Medications 
All treatments that the investigator considers necessary for a subject’s welfare may be 
administered at the discretion of the investigator in keeping with the community standards of 
medical care.  All concomitant medication will be recorded on the case report form (CRF) 
including all prescription, 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 after 30 days after the last dose of trial treatment should be 
recorded for SAEs and ECIs as defined in Section 7.2. 
5.4.2 Prohibited Concomitant Medications 
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 
• Investigational agents other than 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 to modulate symptoms from an 
event of clinical interest of suspected immunologic etiology.  The use of physiologic 
doses of corticosteroids may be approved after consultation with the Sponsor. 
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. 

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The Exclusion Criteria describes other medications which are prohibited in this trial. 
There are no prohibited therapies during the Post-Treatment Follow-up Phase. 
5.5 
Rescue Medications & Supportive Care 
5.5.1 Supportive Care Guidelines 
Subjects should receive appropriate supportive care measures as deemed necessary by the 
treating investigator. Suggested supportive care measures for the management of adverse 
events with potential immunologic etiology are outlined below and in greater detail in the 
ECI guidance document. Where appropriate, these guidelines include the use of oral or 
intravenous 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 or bacterial or viral infection, which might require additional supportive 
care. The treatment guidelines are intended to be applied when the investigator determines 
the events to be related to pembrolizumab.  
Note: if after the evaluation the event is determined not to be related, the investigator is 
instructed to follow the ECI reporting guidance but does not need to follow the treatment 
guidance (as outlined in the ECI guidance document). Refer to Section 5.2.1 for dose 
modification.  
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or 
skin photography as part of evaluation of the event. Suggested conditional procedures, as 
appropriate, can be found in the ECI guidance document. 
 
• Pneumonitis:  
o For Grade 2 events, treat with systemic corticosteroids. When symptoms 
improve to Grade 1 or less, steroid taper should be started and continued over 
no less than 4 weeks. 
o For Grade 3-4 events, immediately treat with intravenous steroids.  
Administer additional anti-inflammatory measures, as needed. 
o Add prophylactic antibiotics for opportunistic infections in the case of 
prolonged steroid administration. 
 
• Diarrhea/Colitis:  
Subjects should be carefully monitored for signs and symptoms of enterocolitis (such 
as diarrhea, abdominal pain, blood or mucus in stool, with or without fever) and of 
bowel perforation (such as peritoneal signs and ileus).   

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o All subjects who experience 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.  For Grade 2 or higher 
diarrhea, consider GI consultation and endoscopy to confirm or rule out 
colitis. 
o For Grade 2 diarrhea/colitis that persists greater than 3 days, administer oral 
corticosteroids.  
o For Grade 3 or 4 diarrhea/colitis that persists > 1 week, treat with 
intravenous steroids followed by high dose oral steroids.   
o When symptoms improve to Grade 1 or less, steroid taper should be started 
and continued over no less than 4 weeks. 
 
 
• Type 1 diabetes mellitus (if new onset, including diabetic ketoacidosis [DKA]) or 
≥ Grade 3 Hyperglycemia, if associated with ketosis (ketonuria) or metabolic 
acidosis (DKA) 
 
o For T1DM or Grade 3-4 Hyperglycemia 
 Insulin replacement therapy is recommended for Type I diabetes 
mellitus and for Grade 3-4 hyperglycemia associated with metabolic 
acidosis or ketonuria.  
 Evaluate patients with serum glucose and a metabolic panel, urine 
ketones, glycosylated hemoglobin, and C-peptide.  
 
• Hypophysitis: 
 
o For Grade 2 events, treat with corticosteroids. When symptoms improve to 
Grade 1 or less, steroid taper should be started and continued over no less than 
4 weeks. Replacement of appropriate hormones may be required as the steroid 
dose is tapered. 
o For Grade 3-4 events, treat with an initial dose of IV corticosteroids followed 
by oral corticosteroids. When symptoms improve to Grade 1 or less, steroid 
taper should be started and continued over no less than 4 weeks. Replacement 
of appropriate hormones may be required as the steroid dose is tapered. 
 
 
• Hyperthyroidism or Hypothyroidism:  
Thyroid disorders can occur at any time during treatment. Monitor patients for 
changes in thyroid function (at the start of treatment, periodically during treatment, 
and as indicated based on clinical evaluation) and for clinical signs and symptoms of 
thyroid disorders. 
 

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o Grade 2 hyperthyroidism events (and Grade 2-4 hypothyroidism): 
 In hyperthyroidism, non-selective beta-blockers (e.g. propranolol) are 
suggested as initial therapy. 
 In hypothyroidism, thyroid hormone replacement therapy, with 
levothyroxine or liothyroinine, is indicated per standard of care. 
o Grade 3-4 hyperthyroidism  
 Treat with an initial dose of IV corticosteroid followed by oral 
corticosteroids. When symptoms improve to Grade 1 or less, steroid 
taper should be started and continued over no less than 4 weeks. 
Replacement of appropriate hormones may be required as the steroid 
dose is tapered. 
 
 
• Hepatic: 
o For Grade 2 events, monitor liver function tests more frequently until 
returned to baseline values (consider weekly). 
 Treat with IV or oral corticosteroids 
o For Grade 3-4 events, treat with intravenous corticosteroids for 24 to 48 
hours.  
o When symptoms improve to Grade 1 or less, a steroid taper should be started 
and continued over no less than 4 weeks. 
 
• Renal Failure or Nephritis: 
o For Grade 2 events, treat with corticosteroids. 
o For Grade 3-4 events, treat with systemic corticosteroids. 
o When symptoms improve to Grade 1 or less, steroid taper should be started and 
continued over no less than 4 weeks.  
 
Management of Infusion Reactions: Signs and symptoms usually develop during or 
shortly after drug infusion and generally resolve completely within 24 hours of 
completion of infusion.  
Table  
Table 4 below shows treatment guidelines for subjects who experience an infusion 
reaction associated with administration of pembrolizumab (MK-3475). 
Table 4 Infusion Reaction Treatment Guidelines 
NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
Grade 1 
Mild reaction; infusion interruption 
not indicated; intervention not 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
None 

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NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
indicated 
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 premedicated 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 premedicated 1.5h 
(± 30 minutes) prior to infusion of 
pembrolizumab (MK-3475) 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. 
 
 
5.6 
Diet/Activity/Other Considerations 
5.6.1 Diet 
Subjects should maintain a normal diet unless modifications are required to manage an AE 
such as diarrhea, nausea or vomiting. 
5.6.2 Contraception 
Pembrolizumab may have adverse effects on a fetus in utero.  Furthermore, it is not known if 
pembrolizumab has transient adverse effects on the composition of sperm. Non-pregnant, 

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non-breast-feeding women may be enrolled if they are willing to use 2 methods of birth 
control or are considered highly unlikely to conceive.  Highly unlikely to conceive is defined 
as 1) surgically sterilized, or 2) postmenopausal (a woman who is ≥45 years of age and has 
not had menses for greater than 1 year will be considered postmenopausal), or 3) not 
heterosexually active for the duration of the study.  The two birth control methods can be 
either two barrier methods or a barrier method plus a hormonal method to prevent pregnancy. 
Subjects should start using birth control from study Visit 1 throughout the study period up to 
120 days after the last dose of study therapy.  
The following are considered adequate barrier methods of contraception: diaphragm, condom 
(by the partner), copper intrauterine device, sponge, or spermicide.  Appropriate hormonal 
contraceptives will include any registered and marketed contraceptive agent that contains an 
estrogen and/or a progestational agent (including oral, subcutaneous, intrauterine, or 
intramuscular agents). 
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.  In order to participate 
in the study they must adhere to the contraception requirement (described above) for the 
duration of the study and during the follow-up period defined in section 7.2.2-Reporting of 
Pregnancy and Lactation to the Sponsor and to Merck.  If there is any question that a subject 
will not reliably comply with the requirements for contraception, that subject should not be 
entered into the study. 
5.6.3 Use in Pregnancy 
If a subject inadvertently becomes pregnant while on treatment with pembrolizumab, 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 the Sponsor and to Merck 
without delay and within 24 hours to the Sponsor and within 2 working days to Merck if the 
outcome is a serious adverse 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 Merck and followed as 
described above and in Section 7.2.2. 
5.6.4 Use in Nursing Women 
It is unknown whether pembrolizumab is excreted in human milk.  Since many drugs are 
excreted in human milk, and because of the potential for serious adverse reactions in the 
nursing infant, subjects who are breast-feeding are not eligible for enrollment. 

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5.7 
Subject Withdrawal/Discontinuation Criteria 
Subjects may withdraw consent at any time for any reason or be dropped from the trial at the 
discretion of the investigator should any untoward effect occur.  In addition, a subject may be 
withdrawn by the investigator or the Sponsor if enrollment into the trial is inappropriate, the 
trial plan is violated, or for administrative and/or other safety reasons.  Specific details 
regarding discontinuation or withdrawal are provided in Section 7.1.3.1 – Other Procedures. 
A subject must be discontinued from the trial for any of the following reasons: 
• The subject or legal representative (such as a parent or legal guardian) withdraws 
consent. 
• Confirmed radiographic disease progression as described in Section 5.7.1 
• Unacceptable adverse experiences as described in Section 7.2.3 
• Intercurrent illness that prevents further administration of treatment 
• Investigator’s decision to withdraw the subject 
• The subject has a confirmed positive serum pregnancy test 
• Noncompliance with trial treatment or procedure requirements 
• The subject is lost to follow-up 
• Completed 24 months of uninterrupted treatment with pembrolizumab or 35 
administrations of study medication, whichever is later. 
Note: 24 months of study medication is calculated from the date of first dose. 
Administrative reasons 
 
The End of Treatment and Follow-up visit procedures are listed in Section 6 (Protocol Flow 
Chart) and Section 7.1.4 (Visit Requirements). After the end of treatment, each subject will 
be followed for 30 days for adverse event monitoring (serious adverse events will be 
collected for 90 days after the end of treatment as described in Section 7.2.3.  Subjects who 
discontinue for reasons other than progressive disease will have post-treatment follow-up for 
disease status until disease progression, initiating a non-study cancer treatment, withdrawing 
consent or becoming lost to follow-up.  After documented disease progression each subject 
will be followed by telephone for overall survival until death, withdrawal of consent, or the 
end of the study, whichever occurs first. 

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5.7.1 Discontinuation of Study Therapy after CR  
Discontinuation of treatment may be considered for subjects who have attained a confirmed 
CR that have been treated for at least 24 weeks with pembrolizumab and had at least two 
treatments with pembrolizumab beyond the date when the initial CR was declared. 
5.8 
Subject Replacement Strategy 
Subjects who do not receive the first 4 cycles of treatment due to toxicity will be replaced. 
5.9 
Clinical Criteria for Early Trial Termination 
Early trial termination will be the result of the criteria specified below: 
1. Quality or quantity of data recording is inaccurate or incomplete 
2. Poor adherence to protocol and regulatory requirements 
3. Incidence or severity of adverse drug reaction in this or other studies indicates a potential 
health hazard to subjects 
4. Plans to modify or discontinue the development of the study drug 
In the event of Merck decision to no longer supply study drug, ample notification will be 
provided so that appropriate adjustments to subject treatment can be made. 
 

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6.0 TRIAL FLOW CHART 
6.1 
Study Flow Chart 
Trial Period: 
Screening Phase 
Treatment Cyclesa 
End of 
Treatment 
Post-Treatment 
Treatment Cycle/Title: 
Pre-
screening 
(Visit 1) 
Main 
Study 
Screening 
(Visit 2) 
1 
2  
3 
4 
To be repeated beyond 8 
cycles 
Discon 
Safety 
Follow-up 
Follow Up 
Visitsb 
Survival 
Follow-
Up 
5 
6 
7 
8 
Scheduling Window (Days): 
 
-28 to -1 
 
± 3 
± 3 
± 3 
± 3 
± 3 
± 3 
± 3 
At time of 
Discon 
30 days 
post 
discon 
Every 12 
weeks post 
discon 
Every 
12 
weeks  
Administrative Procedures 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion Criteria 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics and Medical History 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Prior and Concomitant Medication Review 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Trial Treatment Administration 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Post-study anticancer therapy status 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Survival Status 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Clinical Procedures/Assessments 
Review Adverse Events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Full Physical Examination 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Directed Physical Examination 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Vital Signs and Weight 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
ECOG Performance Status 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Laboratory Procedures/Assessments: analysis performed by LOCAL laboratory 
Pregnancy Test – Urine or Serum β-HCG 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 

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Trial Period: 
Screening Phase 
Treatment Cyclesa 
End of 
Treatment 
Post-Treatment 
Treatment Cycle/Title: 
Pre-
screening 
(Visit 1) 
Main 
Study 
Screening 
(Visit 2) 
1 
2  
3 
4 
To be repeated beyond 8 
cycles 
Discon 
Safety 
Follow-up 
Follow Up 
Visitsb 
Survival 
Follow-
Up 
5 
6 
7 
8 
Scheduling Window (Days): 
 
-28 to -1 
 
± 3 
± 3 
± 3 
± 3 
± 3 
± 3 
± 3 
At time of 
Discon 
30 days 
post 
discon 
Every 12 
weeks post 
discon 
Every 
12 
weeks  
PT/INR and aPTT 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with Differential with platelet count 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Comprehensive Serum Chemistry Panel 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Urinalysisc 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
T3, FT4 and TSH and cortisol c 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Autoimmunity paneld 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
HIV, Hepatitis B and C, Influenzae 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
HLA class I (A, B, C) and II (DR, DQ, DP) 
Testing 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Efficacy Measurements 
Tumor Imagingf 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Tumor Biopsies/Archival Tissue Collection/Correlative Studies Blood 
Archival or Newly Obtained Tissue 
Collectiong 
 
X 
 
 
 
Xh 
 
 
 
 
 
 
 
 
Correlative Studies Blood Collectioni 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
a: treatment cycle is defined as 21 days (3 weeks). Therefore treatment cycles 1, 2, 3, 4, 5, 6, 7, 8, and 9 are defined as starting 
respectively at week 1, 4, 7, 10, 13, 16, 19, 22, and 25. Treatment will be continued every 3 weeks unless there is a reason to 
discontinue. Treatment can be given +/- 3 days of the onset of each treatment cycle.  

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b: follow up every 12 weeks (+/-1 week) following completion or discontinuation  of pembrolizumab infusions until progression or 
patient consent withdrawal or 3 years post-enrollment.  The safety follow-up visit will be 30 days post discontinuation of study agent 
+/- 3 days) 
c: results of the endocrine studies are not required to be back to initiate treatment each cycle. In cases where urinalysis not able to be 
provided treatment could still be administered following discussion with the PI.  
d: autoimmunity panel includes: RF (rheumatoid factor), ANA (antinuclear antibody) and anti-DNA. 
e: include HCV, RNA (qualitative) or Hepatitis C antibody, HBsAg, and HIV type I and type 2 (e.g., HIV-1/-2 antibody screening test 
and evaluation of HIV viral load as needed).  
f: imaging should be performed during week 3 of every 4th treatment cycle while receiving pembrolizumab, i.e. every 12 weeks. For 
example week 12 and 24. If imaging raises concern for pseudo- progression, imaging should be repeated 4-6 weeks later to help 
differentiate between true versus pseudo- progression.  
g: archival if available, or optional new biopsy by incisional or excisional or core needle biopsy if easily accessible metastasis 
(epidermal, dermal, or subcutaneous or palpable lymph node) prior to cycle 1 day 1. Optional incisional or excisional or core needle 
biopsy if easily accessible metastasis (epidermal, dermal, or subcutaneous or palpable lymph node) during week 11 or 12 of the study.  
h: optional tissue biopsy.   
i: correlative blood collection obtained at baseline (2 values), week 4, week 7, week 10, week 13, week 16, week 19, week 22, week 
25, week 34, week 52, week 78, week 104 and/or at end of treatment (EOT), and 30 days post discontinuation.. Samples should be 
obtained prior to pembrolizumab infusion, ideally on the day of the planned infusion. Samples can be obtained +/- 3 days of the start 
of the treatment cycle. Two baseline values will be obtained, one to be drawn the day of first planned pembrolizumab infusion but 
prior to the infusion and the other within 7 days prior to cycle1 day1. 
Blood volumes for melanoma patients: Screening, week 1, week 16 and 30 days post discontinuation up to 80ml; all other visits up to 
40ml. 
Blood volumes for healthy donors: Healthy donors will provide up to 60 ml of blood at 2 time points at least one month apart. 

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7.0 TRIAL PROCEDURES 
7.1 
Trial Procedures 
The Trial Flow Chart - Section 6.0 summarizes the trial procedures to be performed at each 
visit.  Individual trial procedures are described in detail below.  It may be necessary to 
perform these procedures at unscheduled time points if deemed clinically necessary by the 
investigator. 
Furthermore, additional evaluations/testing may be deemed necessary by the Sponsor and/or 
Merck for reasons related to subject safety. In some cases, such evaluation/testing may be 
potentially sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may 
require that additional informed consent be obtained from the subject.  In these cases, such 
evaluations/testing will be performed in accordance with those regulations. 
7.1.1 Administrative Procedures 
7.1.1.1 Informed Consent 
The Investigator must obtain documented consent from each potential subject prior to 
participating in a clinical trial. 
Consent must be documented by the subject’s dated signature or by the subject’s legally 
acceptable representative’s dated signature on a consent form along with the dated signature 
of the person conducting the consent discussion.  
A copy of the signed and dated consent form should be given to the subject before 
participation in the trial. 
The initial informed consent form, any subsequent revised written informed consent form 
and any written information provided to the subject must receive the IRB/ERC’s 
approval/favorable opinion in advance of use. The subject or his/her legally acceptable 
representative should be informed in a timely manner if new information becomes available 
that may be relevant to the subject’s willingness to continue participation in the trial.  The 
communication of this information will be provided and documented via a revised consent 
form or addendum to the original consent form that captures the subject’s dated signature or 
by the subject’s legally acceptable representative’s dated signature. 
Specifics about a trial and the trial population will be added to the consent form template at 
the protocol level.   
The informed consent will adhere to IRB/ERC requirements, applicable laws and regulations 
and Sponsor requirements. 
 

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7.1.1.2 Inclusion/Exclusion Criteria 
All inclusion and exclusion criteria will be reviewed by the investigator or qualified designee 
to ensure that the subject qualifies for the trial.  
7.1.1.3 Medical History 
A medical history will be obtained by the investigator or qualified designee.  Medical history 
will include all active conditions, and any condition diagnosed within the prior 10 years that 
are considered to be clinically significant by the Investigator.  Details regarding the disease 
for which the subject has enrolled in this study will be recorded separately and not listed as 
medical history.   
7.1.1.4 Prior and Concomitant Medications Review 
7.1.1.4.1 Prior Medications 
The investigator or qualified designee will review prior medication use, including any 
protocol-specified washout requirement, and record prior medication taken by the subject 
within 28 days before starting the trial.  Treatment for the disease for which the subject has 
enrolled in this study will be recorded separately and not listed as a prior medication.  
7.1.1.4.2 Concomitant Medications 
The investigator or qualified designee will record medication, if any, taken by the subject 
during the trial.  All medications related to reportable SAEs and ECIs should be recorded as 
defined in Section 7.2. 
7.1.1.5 Disease Details and Treatments 
7.1.1.5.1 Disease Details 
The investigator or qualified designee will obtain prior and current details regarding disease 
status.  
7.1.1.5.2 Prior Treatment Details 
The investigator or qualified designee will review all prior cancer treatments including 
systemic treatments, radiation and surgeries. 
7.1.1.5.3 Subsequent Anti-Cancer Therapy Status 
The investigator or qualified designee will review all new anti-neoplastic therapy initiated 
after the last dose of trial treatment.  If a subject initiates a new anti-cancer therapy within 30 
days after the last dose of trial treatment, the 30 day Safety Follow-up visit must occur before 

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the first dose of the new therapy.  Once new anti-cancer therapy has been initiated, the 
subject will move into the survival follow-up phase.  
7.1.2 Clinical Procedures/Assessments 
7.1.2.1 Adverse Event (AE) Monitoring 
The investigator or qualified designee will assess each subject to evaluate for potential new 
or worsening AEs as specified in the Trial Flow Chart and more frequently if clinically 
indicated.  Adverse experiences will be graded and recorded throughout the study and during 
the follow-up period according to NCI CTCAE Version 4.0 (see Section 11.3).  Toxicities 
will be characterized in terms regarding seriousness, causality, toxicity grading, and action 
taken with regard to trial treatment.  
For subjects receiving treatment with pembrolizumab all AEs of unknown etiology 
associated with pembrolizumab exposure should be evaluated to determine if it is possibly an 
event of clinical interest (ECI) of a potentially immunologic etiology (termed immune-
related adverse events, or irAEs); see the separate ECI guidance document in Appendix 4 
regarding the identification, evaluation and management of potential irAEs.   
Please refer to section 7.2 for detailed information regarding the assessment and recording of 
AEs.   
7.1.2.2 Full Physical Exam 
The investigator or qualified designee will perform a complete physical exam during the 
screening period.  Clinically significant abnormal findings should be recorded as medical 
history.  A full physical exam should be performed during screening,  
7.1.2.3 Directed Physical Exam 
For cycles that do not require a full physical exam per the Trial Flow Chart, the investigator 
or qualified designee will perform a directed physical exam as clinically indicated prior to 
trial treatment administration.   
7.1.2.4 Vital Signs 
The investigator or qualified designee will take vital signs at screening, prior to the 
administration of each dose of trial treatment and at treatment discontinuation as specified in 
the Trial Flow Chart (Section 6.0).  Vital signs should include temperature, pulse, respiratory 
rate, weight and blood pressure.  Height will be measured at screening only. 

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7.1.2.5 Eastern Cooperative Oncology Group (ECOG) Performance Scale 
The investigator or qualified designee will assess ECOG status (see Section 11.1) at 
screening, prior to the administration of each dose of trial treatment, and at discontinuation of 
trial treatment as specified in the Trial Flow Chart.    
7.1.2.6 Tumor Imaging and Assessment of Disease 
Contrast enhanced CT imaging is preferred and to be obtained of chest, abdomen and pelvis 
and should encompass other known areas of metastases. MRI imaging or fusion PET/CT 
imaging can be performed if not able to tolerate CT contrast.  This imaging will be 
performed for baseline assessment between day -28 and day -1.  
A brain MRI will be obtained (contrast enhanced preferred) or if cannot tolerate MRI a CT 
head (contrast enhanced preferred) as baseline assessment between day -28 and day -1.  
Imaging will be repeated during week 3 of every 4th treatment cycle (approximately every 
12th week) while receiving pembrolizumab. The extracranial imaging will be performed 
every 8 weeks in patients with response or stable disease no longer taking pembrolizumab 
due to toxicity for up to 2 years.  
If patient has delay in treatment timing because of toxicity or another reason unrelated to 
study drug such as unrelated medical or personal family event imaging will remain as per 
original schedule with dates based on timing of cycle #1 day#1 (approx. every 12 weeks). 
 
7.1.2.7 Tumor Tissue Collection and Correlative Studies Blood Sampling 
Samples will be stored and processed in the laboratory of Dr. Nina Bhardwaj, Hess Center 
for Science and Medicine, 1470 Madison Avenue 5th floor. Investigators performing these 
assays will be blinded to clinical response data. 
Blood specimens. Blood samples for cell profiling and plasma measurements will be 
collected at baseline (2 values; visit 2 and cycle 1 prior to cycle 1), week 4 (prior to cycle 2), 
week 7 (prior to cycle 3), week 10 (prior to cycle 4), week 13 (prior to cycle 5), week 16 
(prior to cycle 6), week 19 (prior to cycle 7), week 22 (prior to cycle 8), week 25 (prior to 
cycle 9), week 34 (prior to infusion10), week 52, week 78, week 104, at time of 
discontinuation (EOT= End of treatment) and 30 days post discontinuation to assess for (a) 
NK cell exhaustion phenotype; (b) plasma levels of MICA, HMGB1 and CEACAM-1; (c) 
ALC; (d) MDSC frequency and (e) frequency of ICOS+CD4+ and EOMES+ CD8+ T cells.  
NK cells will be analyzed for a set of phenotypic and functional parameters. Their phenotype 
will be assessed by flow cytometry for expression of Tim-3, CEACAM-1, in addition to 
other selected exhaustion markers, IL-2R subunits (alpha, beta, gamma), and a panel of 

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activating and inhibitory NKR (NK cell receptors). To assess functional activity, NK cells 
will be analyzed for cytokine production, cytotoxicity and proliferation.  
An exhausted NK cell phenotype will be characterized by up-regulation of inhibitory 
receptors (Tim-3, CEACAM1 and others), down-regulation of activating receptors (NKG2D, 
NKp46 and others), IL-2R and transcription factors (T-bet and Eomes), and/or loss of IFNγ 
production, proliferation and cytotoxicity. Significant reduction (>30%), compared with a 
mean of healthy donor values, obtained from healthy subjects in this trial and as previously 
reported4, in any one or more of three functional parameters, namely, proliferation, CTL, 
IFNγ production and/or IL-2Receptor expression will constitute the phenotype of “NK cell 
exhaustion”. Our prior data indicate that these parameters generally go hand in hand. These 
studies will be supplemented by CyTOF analysis to validate pathways we find to be 
associated with the exhaustion state Plasma MICA, HMGB1 and CEACAM-1 will be 
monitored by ELISA.  
a) NK cell exhaustion phenotype 
NK cell purification: NK cells isolated from peripheral blood mononuclear cells (PBMCs) 
purified from healthy donor blood (HD; obtained as part of an IRB approved protocol or 
from melanoma donor blood (MD) by Ficoll-Paque Plus centrifugation. NK-cell enrichment 
will be performed by negative selection using Easy-Sep Human NK cell Enrichment Kit 
(StemCell Technologies) according to the manufacturer’s recommendations, obtaining more 
than 95% CD3-CD56+ populations. 
NK Cell Staining and Flow Cytometry Analysis: NK cells from HD and MD will be 
assessed by Flow Cytometry for the activating receptors CD16, NKp30, NKp44, and DNAX 
accessory molecule-1 (DNAM-1), and inhibitory receptors that recognize MHC class I 
molecules and block NK cell-mediated cytotoxicity. They include the Killer cell Ig-like 
Receptor (KIR) family, the Leukocyte immunoglobulin-like Receptor (LILR, CD85) family 
and the family of CD94/NKG2A lectin-like receptors. Expression of checkpoint molecules 
such as Tim-3, CEACAM-1, B7-H3, VISTA, LAG-3, CD96, PD-1, CTLA-4 and TIGIT will 
also be monitored.  Expression patterns of receptors for cytokines (IL-2, IL-12, IL-15 and IL-
18) that mediate NK cell growth, activation and function will be analyzed. Finally, NK cells 
will be assessed by intracellular analysis for expression of the transcription factors T-bet and 
Eomes. 
Flow cytometry analysis will be undertaken at two different points; i) cells freshly isolated 
from blood, and ii) after in vitro O/N stimulation with hrIL-2 (1000U/ml). 
IFNγ production assay: Purified NK cells will be cultured O/N with recombinant human IL-
2 (rhIL-2, 1000U/ml) and after overnight culture, stimulated for 4h in the presence of 1µg/ml 
of rhIL-12 and brefeldin A (10µg/ml, Sigma). Cells are then fixed (1% paraformaldehyde, 

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Electron Microscopy Sciences) and permeabilized  (0.1% saponin, Sigma)). Permeabilized 
cells will be stained for intracellular IFNγ and analyzed by flow cytometry. 
Cytotoxicity Assay: Purified NK cells will be cultured O/N with rhIL-2 (1000U/ml) and 
then stimulated with target cells (K562 cell) at a 5:1 effector/target ratio in combination with 
anti-CD107 antibody (0.5µg/ml) and monensin 1000X (BioLegend). After a 4h incubation, 
CD107 expression on CD56+ cells is quantified by flow cytometry. CD107 is expressed on 
the cell surface as a result of degranulation. 
Proliferation assay: Purified NK cells will be loaded with 2µM CFSE (Life Technologies) 
and cultured in complete media supplemented with 200U/ml of rhIL-2. After 6 days, CFSE 
dilution is analyzed by flow cytometry as a measure of cell proliferation. 
b) Identification of inflammatory pathways that are modulated in exhausted NK cells 
Mass cytometry measurement (CyTOF): 
Isolated PBMCs will be cultured overnight in the presence of 1000U/ml of rhIL-2 and 
complete media. After removal and replacement of media, cells are stimulated with rhIL-2 
(1000U/ml), rhIL-15 (100 ng/ml), rhIL-12 (1ug/ml) and/or anti-CD16 (10ug/ml) for 0, 15, 30 
min-1h. A commercial viability marker, Cisplatin, is added to the culture 5min before 
treatment is completed. Cells will be washed and fixed with 1% PFA for 20min at RT and 
subsequently stained with the appropriate barcode antibody, CD45-115, CD45-141, CD45-
159 and CD45-169 (each barcode will designate a specific time point). Cells are washed 
again and incubated for 10 min at 4C with a cocktail of metal conjugated antibodies 
containing i) anti-CD56, -CD3, -CD4, -CD16 to identify cellular subsets and distinguish NK 
cells; ii) anti-Tim-3, -CEACAM-1, -NKG2D, -NKp46, -DNAM-1, -IL-2R, -KIR3DL1,-
KIR2DL3 to identify cellular subsets of exhausted NK cells.  
Identification of signaling mechanisms that promote exhaustion will be performed by 
intracellular staining. NK cells will be evaluated for intracellular mediators that signal 
activation pathways including STAT and Lck proteins. STAT4 and STAT5 are involved in 
IFNγ production, critically important for early control of tumors, and Lck, which is 
modulated by inhibitory signals mediated by Tim-3 via Bat311. 
 Cells will be permeabilized with 100% cold Methanol for 20min at 4C, washed and stained 
with anti-phospho STAT-4, STAT-5 and anti-phospho-Lck antibodies. Acquisition and 
detection of samples will be done using a CyTOF mass cytometer device in our Immune 
Monitoring Core facility. Data will be analyzed using Cytobank software. NK cells will be 
analyzed by CyTOF prior to beginning treatment, and at two additional time points; week 16 
(prior to cycle 6) and 30 days post discontinuation. 
Transcriptome analysis  
Global changes in gene expression through RNA seq (RNA poly A library and small RNA 
sequencing to target miRNA) using the Illumina platform will be applied to observe and 

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confirm cellular pathway alterations and gene expression level changes from HD and MD 
NK cells during progression of the treatment. RNA from HD and MD enrolled in this trial 
will be isolated from collected whole blood.  
Immune multiplex gene expression 
A multiplex gene expression analysis with 770 genes from 24 different immune cell types 
covering both the adaptive and innate immune response will be performed on RNA isolated 
from HD and MD enrolled in this trial. RNA will be isolated from collected whole blood. 
The analysis and detection of immune-related genes will be done by nCounter PanCancer 
Immune Profiling Panel (Nanostring Technologies). 
cc) Plasma levels of MICA, HMGB1 and CEACAM-1 
ELISA assays: Expression levels of MICA, HMGB1 and CEACAM1 on HD and patients’ 
plasma will be performed using commercially available kits (R&D systems and antibodies-
online, and SIGMA respectively). 
Luminex or equivalent technology: Expression levels of human cytokines on HD and 
patient’s plasma will be analyzed using standard multiplexed Luminex technology (Cytokine 
Human Magnetic 35-Plex Panel for LuminexTM) and/or Olink or equivalent methodology. 
d) Frequency of MDSC; ICOS+CD4+ and EOMES+ CD8+ T cells 
Flow cytometry analysis: Myeloid-derived suppressor cells (MDSC) will be detected by 
flow cytometry and are characterized by CD3+ HLA-DR-/lo, CD11b+, CD33+, CD34+, 
arginase-I+ and ROS+. Circulating ICOS+CD4+ T cells and EOMES+CD8+ T cells will also 
be assessed by Flow Cytometry. 
e) Tumor-infiltrating NK cells in tissue samples 
Archived tumor (at least 10 unstained slides or tissue block) will be requested at baseline, if 
available. Subjects may undergo optional tumor biopsies of easily accessible metastatic 
deposits, if present (epidermal, dermal, subcutaneous, palpable lymph node), by incisional, 
excisional, or core needle biopsy prior to cycle 1 day 1. Optional incisional, excisional, or 
core needle biopsy of easily accessible metastasis (epidermal, dermal, or subcutaneous or 
palpable lymph node) may be performed during week 11 or 12 of the study.  
Depending on tumor tissue availability, tumor biopsy specimens will be subdivided for 
paraffin embedding and for snap freezing for immuno-fluorescence analysis. If sufficient 
material is available, fresh tumor will be digested to obtain cell suspensions for flow 
cytometry analysis, and immune functional assays.  
Immunohistochemistry (IHC): The following monoclonal antibodies will be used for 
detecting NK cells in tissue specimens: CD56, CD16 and CD57. Expression of NK cell 

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receptors and analysis of exhaustion will be performed by analyzing NKp30, NKp46, Tim-3 
and CEACAM1 expression and other relevant exhaustion markers. A multiplex 
immunofluorescence assay designed at Mount Sinai by Dr. Sacha Gnjatic, co-Director of the 
Immune Monitoring Core will be used for these assessments.  
Single-cell isolation from tissue samples: Similar assays as described in a) will be 
performed on NK cells isolated from fresh tissue, where possible. Single-cell isolation will 
be performed on collagenase treated tissue and NK cells selected by sorting.  
 
7.1.3 Laboratory Procedures/Assessments 
Details regarding specific laboratory procedures/assessments (Hematology, Chemistry and 
Urinalysis) to be performed in this trial are provided in Table 5. 
 

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Table 5 - Laboratory Tests 
Hematology 
Chemistry 
Urinalysis 
Other 
Hematocrit 
Albumin 
Blood 
Serum β-human chorionic gonadotropin† 
Hemoglobin 
Alkaline phosphatase 
Glucose 
  (β-hCG)† 
Platelet count 
Alanine aminotransferase (ALT) 
Protein 
PT (INR) 
WBC (total and differential) 
Aspartate aminotransferase (AST) 
Specific gravity  
aPTT 
Red Blood Cell Count 
Lactate dehydrogenase (LDH) 
Microscopic exam (If abnormal) 
results are noted 
T3 
Absolute Neutrophil Count 
Carbon Dioxide 
Urine pregnancy test † 
Free tyroxine (T4)  
Absolute Lymphocyte Count 
  (CO2 or biocarbonate) 
 
Thyroid stimulating hormone (TSH) 
 
Uric Acid 
 
Cortisol 
 
Calcium 
 
 
 
Chloride 
 
Blood for correlative studies 
 
Glucose 
 
 Hepatitis HBsAg 
 
Phosphorus 
 
 Hepatitis C (HCV RNA) or Hepatitis C 
antibody 
 
Potassium   
 
 HIV 
 
Sodium 
 
 Antinuclear antibody (ANA) 
 
Magnesium 
 
 Rheumatoid factor (RF) 
 
Total Bilirubin 
 
 Anti-DNA 
 
Direct Bilirubin (If total bilirubin is 
elevated above the upper limit of 
normal) 
 
 
 
Total protein 
 
 
 
Blood Urea Nitrogen 
 
 
† Perform on women of childbearing potential only. If urine pregnancy results cannot be confirmed as negative, a serum pregnancy test will be required.   
 
 

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Protocol/Amendment No.: 01/ 06          
 
 
 
 
 
 
 
 
 
44
Laboratory tests for screening should be performed within 10 days prior to the first dose of 
treatment.  After Cycle 1, pre-dose laboratory procedures can be conducted up to 72 hours 
prior to dosing.  Results must be reviewed by the investigator or qualified designee and found 
to be acceptable prior to each dose of trial treatment.  
7.1.3.1 Withdrawal/Discontinuation 
When a subject discontinues/withdraws prior to trial completion, all applicable activities 
scheduled for the final trial visit should be performed at the time of discontinuation.  Any 
adverse events which are present at the time of discontinuation/withdrawal should be 
followed in accordance with the safety requirements outlined in Section 7.2 - Assessing and 
Recording Adverse Events.  Subjects who a) attain a CR or b) complete 24 months of 
treatment with pembrolizumab may discontinue treatment. After discontinuing treatment 
following assessment of CR, these subjects should return to the site for a Safety Follow-up 
Visit (described in Section 7.1.4) and then proceed to the Follow-Up Period of the study 
(described in Section 7.1.4). 
7.1.4 Visit Requirements 
Visit requirements are outlined in Section 6.0 - Trial Flow Chart. Specific procedure-related 
details are provided above in Section 7.1 - Trial Procedures. 
Screening Period: from day-28 to day-1. See Section 6 (Study Flow Chart) for details of 
screening items 
Treatment Period: every 3 weeks starting cycle1 day 1 with window +/- 3 days.  
Post-Treatment Visits: follow up every 12 weeks (+/-1 week) following completion or 
discontinuation of pembrolizumab infusions until progression, patient consent withdrawal, or 
3 years post-enrollment.   
Safety Follow-Up Visit 
The mandatory Safety Follow-Up Visit should be conducted approximately 30 days after the 
last dose of trial treatment or before the initiation of a new anti-cancer treatment, whichever 
comes first. All AEs that occur prior to the Safety Follow-Up Visit should be recorded.  
Subjects with an AE of Grade > 1 will be followed until the resolution of the AE to Grade 0-
1 or until the beginning of a new anti-neoplastic therapy, whichever occurs first. SAEs that 
occur within 90 days of the end of treatment or before initiation of a new anti-cancer 
treatment should also be followed and recorded. 
 

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Protocol/Amendment No.: 01/ 06          
 
 
 
 
 
 
 
 
 
45
Follow-up Visits 
Subjects who discontinue trial treatment for a reason other than disease progression will 
move into the Follow-Up Phase and should be assessed every 8 weeks (± 7 days) by 
radiologic imaging to monitor disease status. Every effort should be made to collect 
information regarding disease status until the start of new anti-neoplastic therapy, disease 
progression, death, end of the study. Information regarding post-study anti-neoplastic 
treatment will be collected if new treatment is initiated. 
Survival Follow-up 
Once a subject experiences confirmed disease progression or starts a new anti-cancer 
therapy, the subject moves into the survival follow-up phase and should be contacted by 
telephone every 12 weeks to assess for survival status until death, withdrawal of consent, or 
the end of the study, whichever occurs first.  
 
7.2 
Assessing and Recording Adverse Events 
An adverse event is defined as any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product and which does not necessarily 
have to have a causal relationship with this treatment. An adverse event can therefore be any 
unfavorable and unintended sign (including an abnormal laboratory finding, for example), 
symptom, or disease temporally associated with the use of a medicinal product or protocol-
specified procedure, whether or not considered related to the medicinal product or protocol-
specified procedure.  Any worsening (i.e., any clinically significant adverse change in 
frequency and/or intensity) of a preexisting condition that is temporally associated with the 
use of Pembrolizumab, is also an adverse event. 
Changes resulting from normal development that do not vary significantly in frequency or 
severity from expected levels are not to be considered adverse events.  Examples of this may 
include, but are not limited to, onset of menopause occurring at a physiologically appropriate 
time. 
Adverse events may occur during the course of the use of Pembrolizumab in clinical trials or 
within the follow-up period specified by the protocol, or prescribed in clinical practice, from 
overdose (whether accidental or intentional), from abuse and from withdrawal. 
Adverse events may also occur in screened subjects during any pre-allocation baseline period 
as a result of a protocol-specified intervention, including washout or discontinuation of usual 
therapy or a procedure. 
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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46
All adverse events will be recorded from the time the consent form is signed through 30 days 
following cessation of treatment and at each examination on the Adverse Event case report 
forms/worksheets. The reporting timeframe for adverse events meeting any serious criteria is 
described in section 7.2.3.1. 
7.2.1 Definition of an Overdose for This Protocol and Reporting of Overdose to the 
Sponsor and to Merck 
For purposes of this trial, an overdose of pembrolizumab will be defined as any dose of 1,000 
mg or greater (≥5 times the indicated dose).  No specific information is available on the 
treatment of overdoses of pembrolizumab. Appropriate supportive treatment should be 
provided if clinically indicated. In the event of overdose, the subject should be observed 
closely for signs of toxicity.  Appropriate supportive treatment should be provided if 
clinically indicated. 
If an adverse event(s) is associated with (“results from”) the overdose of pembrolizumab, the 
adverse event(s) is reported as a serious adverse event, even if no other seriousness criteria 
are met. 
If a dose of pembrolizumab meeting the protocol definition of overdose is taken without any 
associated clinical symptoms or abnormal laboratory results, the overdose is reported as a 
non-serious Event of Clinical Interest (ECI), using the terminology “accidental or intentional 
overdose without adverse effect.” 
All overdoses with and without an adverse event must be reported within 24 hours to the 
Sponsor and within 2 working days to Merck Global Safety. (Attn: Worldwide Product 
Safety; FAX 215 993-1220) 
7.2.2 Reporting of Pregnancy and Lactation to the Sponsor and to Merck 
Although pregnancy and lactation are not considered adverse events, it is the responsibility of 
investigators or their designees to report any pregnancy or lactation in a subject 
(spontaneously reported to them), including the pregnancy of a male subject's female partner 
that occurs during the trial or within 120 days of completing the trial, or 30 days following 
cessation of treatment if the subject initiates new anticancer therapy, whichever is earlier.  
All subjects and female partners of male subjects who become pregnant must be followed to 
the completion/termination of the pregnancy.  Pregnancy outcomes of spontaneous abortion, 
missed abortion, benign hydatidiform mole, blighted ovum, fetal death, intrauterine death, 
miscarriage and stillbirth must be reported as serious events (Important Medical Events).  If 
the pregnancy continues to term, the outcome (health of infant) must also be reported. 
Such events must be reported within 24 hours to the Sponsor and within 2 working days to 
Merck Global Safety. (Attn: Worldwide Product Safety; FAX 215 993-1220) 

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Protocol/Amendment No.: 01/ 06          
 
 
 
 
 
 
 
 
 
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7.2.3 Immediate Reporting of Adverse Events to the Sponsor and to Merck 
7.2.3.1 Serious Adverse Events 
A serious adverse event is any adverse event occurring at any dose or during any use of 
pembrolizumab that: 
• Results in death; 
• Is life threatening; 
• Results in persistent or significant disability/incapacity; 
• Results in or prolongs an existing inpatient hospitalization; 
• Is a congenital anomaly/birth defect; 
• Is a new cancer (that is not a condition of the study); 
• Is associated with an overdose; 
• Is an other important medical event  
Refer to Table 6 for additional details regarding each of the above criteria. 
Any serious adverse event, or follow up to a serious adverse event, including death due to 
any cause other than progression of the cancer under study that occurs to any subject from 
the time the consent is signed through 90 days following cessation of treatment, or the 
initiation of new anti-cancer therapy, whichever is earlier, whether or not related to 
pembrolizumab, must be reported within 24 hours to the Sponsor and within 2 working days 
to Merck Global Safety.  
Non-serious Events of Clinical Interest will be forwarded to Merck Global Safety and will be 
handled in the same manner as SAEs.  
Additionally, any serious adverse event, considered by an investigator who is a qualified 
physician to be related to pembrolizumab that is brought to the attention of the investigator at 
any time outside of the time period specified in the previous paragraph also must be reported 
immediately to the Sponsor and to Merck. 
SAE reports and any other relevant safety information are to be forwarded to the Merck 
Global Safety facsimile number:  +1-215-993-1220 
A copy of all 15 Day Reports and Annual Progress Reports is submitted as required by FDA, 
European Union (EU), Pharmaceutical and Medical Devices agency (PMDA) or other local 
regulators. Investigators will cross-reference this submission according to local regulations to 
the Merck Investigational Compound Number (IND, CSA, etc.) at the time of submission.  
Additionally investigators will submit a copy of these reports to Merck & Co., Inc. (Attn: 
Worldwide Product Safety; FAX 215 993-1220) at the time of submission to FDA. 
All subjects with serious adverse events must be followed up for outcome. 

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Protocol/Amendment No.: 01/ 06          
 
 
 
 
 
 
 
 
 
48
7.2.3.2 Events of Clinical Interest 
Selected non-serious and serious adverse events are also known as Events of Clinical Interest 
(ECI) and must be recorded as such on the Adverse Event case report forms/worksheets and 
reported within 24 hours to the Sponsor and within 2 working days to Merck Global Safety 
(Attn: Worldwide Product Safety; FAX 215 993-1220). Events of clinical interest for this 
trial include: 
1.  an overdose of pembrolizumab, as defined in Section 7.2.1 - Definition of an Overdose for 
This Protocol and Reporting of Overdose to the Sponsor, that is not associated with clinical 
symptoms or abnormal laboratory results.  
2.  an elevated AST or ALT lab value that is greater than or equal to 3X the upper limit of 
normal and an elevated total bilirubin lab value that is greater than or equal to 2X the upper 
limit of normal and, at the same time, an alkaline phosphatase lab value that is less than 2X 
the upper limit of normal, 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.  The trial site guidance for 
assessment and follow up of these criteria can be found in the MK-3475 Investigator 
Brochure. 
3. Additional adverse events: 
 
A separate guidance document has been provided entitled “Investigator’s Brochure.  This 
document is provided with this protocol and provides guidance regarding identification, 
evaluation and management of ECIs and irAEs.   
 
ECIs (both non-serious and serious adverse events) identified in this guidance document 
from the date of first dose through 90 days following cessation of treatment, or 30 days 
after the initiation of a new anticancer therapy, whichever is earlier, need to be reported 
within 24 hours to the Sponsor and within 2 working days to Merck Global Safety (Attn: 
Worldwide Product Safety; FAX 215 993-1220), regardless of attribution to study 
treatment, consistent with standard SAE reporting guidelines. 
 
Subjects should be assessed for possible ECIs prior to each dose.  Lab results should be 
evaluated and subjects should be asked for signs and symptoms suggestive of an 
immune-related event.  Subjects who develop an ECI thought to be immune-related 
should have additional testing to rule out other etiologic causes.  If lab results or 
symptoms indicate a possible immune-related ECI, then additional testing should be 
performed to rule out other etiologic causes.  If no other cause is found, then it is 
assumed to be immune-related. 

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Protocol/Amendment No.: 01/ 06          
 
 
 
 
 
 
 
 
 
49
 
7.2.4 Evaluating Adverse Events 
An investigator who is a qualified physician will evaluate all adverse events according to the 
NCI Common Terminology for Adverse Events (CTCAE), version 4.0. Any adverse event 
which changes CTCAE grade over the course of a given episode will have each change of 
grade recorded on the adverse event case report forms/worksheets. 
All adverse events regardless of CTCAE grade must also be evaluated for seriousness. 

Product: Pembrolizumab 
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Table 6 - Evaluating Adverse Events 
An investigator who is a qualified physician, will evaluate all adverse events as to: 
V4.0 
CTCAE 
Grading 
Grade 1 
Mild; asymptomatic or mid symptoms; clinical or diagnostic observations only; intervention not indicated. 
 
Grade 2 
Moderate; minimal, local or noninvasive intervention indicated; limiting age-appropriate instrumental ADL. 
 
Grade 3 
Severe or medically significant but not immediately life-threatening; hospitalization or prolongation or hospitalization indicated; 
disabling; limiting self-care ADL. 
 
Grade 4 
Life threatening consequences; urgent intervention indicated. 
 
Grade 5 
Death related to AE 
Seriousness 
A serious adverse event is any adverse event occurring at any dose or during any use of pembrolizumab that: 
 
†Results in death; or 
 
†Is life threatening; or places the subject, in the view of the investigator, at immediate risk of death from the event as it occurred (Note: This does not include an 
adverse event that, had it occurred in a more severe form, might have caused death.); or 
 
†Results in a persistent or significant disability/incapacity (substantial disruption of one’s ability to conduct normal life functions); or 
 
†Results in or prolongs an existing inpatient hospitalization (hospitalization is defined as an inpatient admission, regardless of length of stay, even if the 
hospitalization is a precautionary measure for continued observation. (Note: Hospitalization [including hospitalization for an elective procedure] for a preexisting 
condition which has not worsened does not constitute a serious adverse event.); or 
 
†Is a congenital anomaly/birth defect (in offspring of subject taking the product regardless of time to diagnosis);or 
 
Is a new cancer; (that is not a condition of the study) or 
 
Is an overdose (whether accidental or intentional).  Any adverse event associated with an overdose is considered a serious adverse event. An overdose that is not 
associated with an adverse event is considered a non-serious event of clinical interest and must be reported within 24 hours. 
 
Other important medical events that may not result in death, not be life threatening, or not require hospitalization may be considered a serious adverse event when, 
based upon appropriate medical judgment, the event may jeopardize the subject and may require medical or surgical intervention to prevent one of the outcomes 
listed previously (designated above by a †). 
Duration 
Record the start and stop dates of the adverse event. If less than 1 day, indicate the appropriate length of time and units 
Action taken  
Did the adverse event cause pembrolizumab to be discontinued? 
Relationship to 
Pembrolizumab  
Did pembolizumab cause the adverse event? The determination of the likelihood that pembrolizumab caused the adverse event will be provided by an investigator 
who is a qualified physician. The investigator’s signed/dated initials on the source document or worksheet that supports the causality noted on the AE form, ensures 
that a medically qualified assessment of causality was done. This initialed document must be retained for the required regulatory time frame. The criteria below are 
intended as reference guidelines to assist the investigator in assessing the likelihood of a relationship between the test drug and the adverse event based upon the 
available information.  
The following components are to be used to assess the relationship between pembrolizumab and the AE; the greater the correlation with the components and 
their respective elements (in number and/or intensity), the more likely pembrolizumab caused the adverse event (AE): 
 
Exposure 
Is there evidence that the subject was actually exposed to pembrolizumab such as: reliable history, acceptable compliance assessment (pill 
count, diary, etc.), expected pharmacologic effect, or measurement of drug/metabolite in bodily specimen? 
 
Time Course 
Did the AE follow in a reasonable temporal sequence from administration of pembrolizumab?  
Is the time of onset of the AE compatible with a drug-induced effect (applies to trials with investigational medicinal product)? 
 
Likely Cause 
Is the AE not reasonably explained by another etiology such as underlying disease, other drug(s)/vaccine(s), or other host or environmental 
factors 

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Relationship 
The following components are to be used to assess the relationship between the test drug and the AE: (continued) 
to 
Pembrolizumab 
(continued)  
Dechallenge 
Was pembrolizumab discontinued or dose/exposure/frequency reduced? 
If yes, did the AE resolve or improve? 
If yes, this is a positive dechallenge. If no, this is a negative dechallenge. 
(Note: This criterion is not applicable if: (1) the AE resulted in death or permanent disability; (2) the AE resolved/improved despite continuation 
of pembrolizumab; or (3) the trial is a single-dose drug trial); or (4) pembrolizumab(s) is/are only used one time.) 
 
Rechallenge 
Was the subject re-exposed to pembrolizumab in this study? 
If yes, did the AE recur or worsen? 
If yes, this is a positive rechallenge. If no, this is a negative rechallenge. 
(Note: This criterion is not applicable if: (1) the initial AE resulted in death or permanent disability, or (2) the trial is a single-dose drug trial); or 
(3) pembrolizumab(s) is/are used only one time). 
NOTE: IF A RECHALLENGE IS PLANNED FOR AN ADVERSE EVENT WHICH WAS SERIOUS AND WHICH MAY HAVE BEEN 
CAUSED BY PEMBROLIZUMAB, OR IF REEXPOSURE TO PEMBROLIZUMAB POSES ADDITIONAL POTENTIAL SIGNIFICANT 
RISK TO THE SUBJECT, THEN THE RECHALLENGE MUST BE APPROVED IN ADVANCE BY THE U.S. CLINICAL MONITOR AS 
PER DOSE MODIFICATION GUIDELINES IN THE PROTOCOL. 
 
Consistency 
with Trial 
Treatment 
Profile 
Is the clinical/pathological presentation of the AE consistent with previous knowledge regarding pembrolizumab or drug class pharmacology or 
toxicology? 
The assessment of relationship will be reported on the case report forms /worksheets by an investigator who is a qualified physician according to his/her best clinical judgment, including 
consideration of the above elements. 
Record one of the following 
Use the following scale of criteria as guidance (not all criteria must be present to be indicative of a pembrolizumab relationship). 
Yes, there is a reasonable 
possibility of Pembrolizumab 
relationship. 
There is evidence of exposure to pembrolizumab.  The temporal sequence of the AE onset relative to the administration of pembrolizumab is 
reasonable.  The AE is more likely explained by pembrolizumab than by another cause. 
No, there is not a reasonable 
possibility of Pembrolizumab 
relationship 
Subject did not receive pembrolizumab OR temporal sequence of the AE onset relative to administration of pembrolizumab is not reasonable 
OR there is another obvious cause of the AE.  (Also entered for a subject with overdose without an associated AE.) 

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Protocol/Amendment No.: 01/ 06  
 
 
  
 
  
 
 
 
52
7.2.5 Sponsor Responsibility for Reporting Adverse Events 
All Adverse Events will be reported to regulatory authorities, IRB/IECs and the principal 
investigator in accordance with all applicable global laws and regulations.  
Each serious adverse event must be reported to the Principal Investigator within 2 business 
days of learning of the occurrence by Fax or email. In the event that the study team does not 
become aware of the serious adverse event immediately (e.g., participant sought treatment 
elsewhere), the study team is to report the event within 2 business days after learning of it 
and document the time of his or her first awareness of the adverse event. Report serious 
adverse events by email or facsimile to:  
 
Nina Bhardwaj MD PhD fax: 646-537-9571 
email:  nina.bhardwaj@mssm.edu  
(Telephone: 212-824-8427) 
 
Within the following 7 business days, the study team must provide follow-up information on 
the serious adverse event. Follow-up information should describe whether the event has 
resolved or continues, if and how the event was treated, and whether the participant will 
continue or discontinue study participation. 
 
Also, all AEs must be reported the IRB according to ISMMS policies. 
 
 
8.0  STATISTICAL ANALYSIS PLAN 
8.1 
Primary Objective 
The primary endpoint is to correlate changes in NK cell exhaustion phenotype with the 
clinical efficacy (best overall response) of pembrolizumab treatment in patients with 
unresectable stage III or IV melanoma. We define NK cell exhaustion being present based on 
reductions of three functional parameters, namely, cytotoxicity, IFNγ production, and 
proliferation. Blood NK cells from melanoma patients develop a functional profile with 
progressive exhaustion from stages I to IV. The exhaustion profile for stages I, II, and III/IV 
melanoma based on mean cytotoxicity is 25%, 22%, and 14%, respectively. The exhaustion 
profile for stage III/IV melanoma shows a mean cytotoxicity of only 14% and a standard 
deviation of 9% (Supplementary Table 1). We expect to see a clinical response rate of 27% 
for pembrolizumab. We therefore estimate that 25-30 patients will respond on 
pembrolizumab and 70-75 patients will not respond. In order to determine if clinical response 
correlates with an improved NK cell function, the % cytotoxicity will be compared between 
responding and non-responding stage III/IV patients. A total of 100 patients (25 responding 
on pembrolizumab and 75 non-responding) would allow detection of a difference of 6% 
between the mean cytotoxity percentages for responding and non-responding patients with 
0.80 power for a two-sided test at the 0.05 level. This difference of 6% (20% for responding 

Product: Pembrolizumab 
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53
patients and 14% for non-responders) corresponds to having a 20% mean cytoxicity for 
responding stage III/IV patients that is close to the expected cytotoxicity for stage II patients 
(mean cytotoxity 22%). This analysis will also be undertaken for IFNγ production and 
proliferation. 
8.2 
Secondary Endpoints 
The first secondary objective is to identify biomarkers associated with a reversal of NK cell 
exhaustion in vivo following treatment with pembrolizumab. A reversal of NK cell 
exhaustion will be defined as reversal of at least one parameter of exhaustion (responsiveness 
to IL-2, IFNg production, and CTL activity). Patients with a reversal of NK exhaustion will 
be compared to patients without a reversal with respect to biomarkers such as HMGB-1, 
CEACAM-1, and MICA. The two groups of patients will be compared over all time points of 
measured plasma levels of HMGB-1, CEACAM-1, and MICA by a repeated measure mixed 
model analysis. This analysis takes into account the correlation between repeated 
measurements obtained on the same patient. A goal here would be to determine whether 
patients with and without a reversal of NK cell exhaustion differ in the pattern of variation 
over time in MICA, HMGB-1, and CEACAM-1 plasma levels. All assumptions to statistical 
procedures will be thoroughly evaluated and transformation of the data will be implemented 
if appropriate. Several approaches will be considered to address the issue of missing data. A 
non-ignorable pattern-mixture model for dropouts will be first proposed. A dropout indicator 
will be included as a covariate in the model. This pattern-mixture model incorporates reason 
for missingness and is most appropriate in this clinical trial setting.  
Other secondary objectives include correlation of changes in NK cell exhaustion phenotype 
with one year survival (and 6 month PFS) following pembrolizumab treatment. Survival (and 
PFS) will be compared for patients with reversal of NK exhaustion and patients without 
reversal by the logrank test. 
 8.3 
Stopping Rules 
Stopping rules are not applicable as Pembrolizumab is routinely used as standard of care 
treatment for stage IV melanoma patients. Toxicities are largely autoimmune and well 
defined from the clinical trials. We are using a flat dose here instead of the FDA approved 
2mg/kg but the flat dose falls within the same dosing range and much higher dosing was used 
in the phase I studies. 
 

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54
9.0 LABELING, 
PACKAGING, 
STORAGE 
AND 
RETURN 
OF 
CLINICAL 
SUPPLIES 
9.1 
Investigational Product  
The investigator shall take responsibility for and shall take all steps to maintain appropriate 
records and ensure appropriate supply, storage, handling, distribution and usage of 
investigational product in accordance with the protocol and any applicable laws and 
regulations. 
Clinical Supplies will be provided by Merck in one of two forms, as summarized in Table 7. 
Table 7 - Product Descriptions 
Product Name & Potency 
Dosage Form 
Pembrolizumab 50 mg 
Lyophilized Powder for Injection 
Pembrolizumab 100 mg/ 4mL 
Solution for Injection 
 
9.2 
Packaging and Labeling Information 
Clinical supplies will be affixed with a clinical label in accordance with regulatory 
requirements. 
9.3 
Clinical Supplies Disclosure 
This trial is open-label; therefore, the subject, the trial site personnel, the Sponsor and/or 
designee are not blinded to treatment. Drug identity (name, strength) is included in the label 
text; random code/disclosure envelopes or lists are not provided. 
9.4 
Storage and Handling Requirements 
Clinical supplies must be stored in a secure, limited-access location under the storage 
conditions specified on the label.   
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. 

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Protocol/Amendment No.: 01/ 06  
 
 
  
 
  
 
 
 
55
9.5 
Returns and Reconciliation 
The investigator, or designee, is responsible for keeping accurate records of the clinical 
supplies received from Merck, 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 institutional 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. 
10.0 ADMINISTRATIVE AND REGULATORY DETAILS 
10.1 
Compliance 
Protocol Review and Amendments: 
This protocol, the proposed informed consent and all forms of participant information 
related to the study (e.g., advertisements used to recruit participants) and any other 
necessary documents must be submitted, reviewed and approved by a properly 
constituted IRB.  
Any changes made to the protocol must be submitted as amendments and must be 
approved by the IRB prior to implementation. Any changes in study conduct must be 
reported to the IRB. The ISMMS Principal Investigator or delegate will disseminate 
protocol amendment information to all participating investigators.  
All decisions of the IRB concerning the conduct of the study must be made in writing. 
 
Informed Consent: 
All participants must be provided a consent form describing this study and providing 
sufficient information for participants to make an informed decision about their 
participation in this study. The formal consent of a participant, using the IRB approved 
consent form, must be obtained before the participant is involved in any study-related 
procedure. The consent form must be signed and dated by the participant or the 
participant’s legally authorized representative, and by the person obtaining the consent. 
The participant must be given a copy of the signed and dated consent document. The 
original signed copy of the consent document must be retained in the medical record or 
research file. 
 
Ethics and Good Clinical Practice (GCP): 
This study is to be conducted according to the following considerations, which represent 
good and sound research practice:  
 

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56
E6 Good Clinical Practice: Consolidated Guidance 
www.fda.gov/downloads/RegulatoryInformation/Guidances/UCM129515.pdf  
US Code of Federal Regulations (CFR) governing clinical study conduct and ethical 
principles that have their origin in the Declaration of Helsinki  
Title 21 Part 11 – Electronic Records; Electronic Signatures 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr11_02.html  
Title 21 Part 50 – Protection of Human Subjects 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr50_02.html  
Title 21 Part 54 – Financial Disclosure by Clinical Investigators 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr54_02.html  
Title 21 Part 56 – Institutional Review Boards 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr56_02.html  
Title 21 Part 312 – Investigational New Drug Application 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr312_02.html  
 
State laws 
 
 
ISMMS research policies and procedures 
 
It is understood that deviations from the protocol should be avoided, except when 
necessary to eliminate an immediate hazard to a research participant. In such case, the 
deviation must be reported to the IRB according to the local reporting policy. 
 
 
Study Documentation  
 
The investigator must prepare and maintain adequate and accurate case histories 
designed to record all observations and other data pertinent to the study for each research 
participant. This information enables the study to be fully documented and the study data 
to be subsequently verified.  
Original source documents supporting entries in the case report forms include but are not 
limited to hospital records, clinical charts, laboratory and pharmacy records, recorded 
data from automated instruments, microfiches, photographic negatives, microfilm or 
magnetic media, and/or x-rays.  
 
Records Retention  
 

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57
They will be retained for a minimum of three years following withdrawal of all subjects 
from the study. All study-related documents must be retained for the maximum period 
required by applicable federal regulations and guidelines or institutional policies. 
 
10.2 
Compliance with Trial Registration and Results Posting Requirements 
Under the terms of the Food and Drug Administration Modernization Act (FDAMA) and 
the Food and Drug Administration Amendments Act (FDAAA), the Sponsor of the trial 
is solely responsible for determining whether the trial and its results are subject to the 
requirements 
for 
submission 
to 
the 
Clinical 
Trials 
Data 
Bank, 
http://www.clinicaltrials.gov.  Information posted will allow subjects to identify 
potentially appropriate trials for their disease conditions and pursue participation by 
calling a central contact number for further information on appropriate trial locations and 
trial site contact information.     
10.3 
Quality Management System 
Data Management:  
The Study team and the Mount Sinai Clinical Trial Office (CCTO) will collect, manage, 
and monitor data for this study.  The principal investigator will assess response rate data.  
 
ERAP will be used for case report forms and CRFs reviewed by study investigator        
for accuracy. Adverse events will be monitored and if unexpected but related SAEs or 
unexpected but related high grade (grade 3 or higher) toxicities felt at least possibly 
related to study drug are observed, the investigators will convene in person or by 
telephone or email to discuss. 
Monitoring: 
This study will be monitored by an independent monitor. The purpose of these audits or 
inspections is to examine study-related activities and documents to determine whether 
these activities were conducted and data were recorded, analyzed, and accurately 
reported in accordance with the protocol, institutional policy, Good Clinical Practice 
(GCP), and any applicable regulatory requirements.  
All data will be monitored for timeliness of submission, completeness, and adherence to 
protocol requirements. Monitoring will begin at the time of participant registration and 
will continue throughout protocol performance and completion.  
 
 
 

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11.0 
APPENDICES 
11.1 
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. Ambulatory and capable of all self-care, 
but unable to carry out any work activities. Up and about more than 
50% of waking hours. 
3 
In bed >50% of the time. Capable of only limited self-care, confined 
to bed or chair more than 50% of waking hours. 
4 
100% bedridden. Completely disabled. Cannot carry on any self-
care. Totally confined to bed or chair. 
5 
Dead. 
* 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. 
 
11.2 
RECIST 1.1 Evaluation criteria 
Measurable lesions must have a longest diameter of ≥ 10 mm on CT with a slice thickness of 
≤5mm (oralongestdiameterof≥20mmon nonhelical CT with a slice thickness of > 10 mm) or 
a longest diameter of ≥ 20 mm on chest radiography. 
Nonmeasurable lesions include other lesions that do not meet the criteria as measurable 
lesions, such as small lesions with a longest diameter of < 10 mm, skeletal metastases 
without a soft-tissue component, ascites, pleural effusion, lymphangitic spread of tumor, 
leptomeningeal disease, inflammatory breast disease, cystic or necrotic lesions, lesions in an 
irradiated area, and an abdominal mass not confirmed by imaging. 
The number of target lesions to be assessed is two per organ and a maximum of five target 
lesions total. 
Lymph nodes with a short axis of ≥ 15 mm are considered measurable and assessable as 
target lesions, and the short-axis measurement should be included in the sum of target lesion 
measurements in the calculation of tumor response as opposed to the longest axis used for 
measurements of other target lesions. Lymph nodes with a short axis of < 10 mm are de- 

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fined as “nonpathologic”. All other pathologic nodes-that is, those with a short axis of ≥ 10 
mm but < 15 mm—should be considered nontarget lesions. 
11.3 
Common Terminology Criteria for Adverse Events V4.0 (CTCAE) 
The descriptions and grading scales found in the revised NCI Common Terminology Criteria 
for Adverse Events (CTCAE) version 4.0 will be utilized for adverse event reporting. 
(http://ctep.cancer.gov/reporting/ctc.html) 
11.4 
Response Evaluation Criteria according to Immune-Related Response Criteria – 
iRC 
Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: 
Immune-Related Response Criteria 
 
At the baseline tumor assessment, the sum of the products of the two largest perpendicular 
diameters (SPD) of all index lesions (five lesions per organ, up to 10 visceral lesions and five 
cutaneous index lesions) is calculated. At each subsequent tumor assessment, the SPD of the 
index lesions and of new, measurable lesions (≥5 x 5 mm; up to 5 new lesions per organ: 5 
new cutaneous lesions and 10 visceral lesions) are added together to provide the total tumor 
burden: 
 
Tumor Burden = SPD index lesions + SPD new, measurable lesions 
 
At each tumor assessment, the response in index and new, measurable lesions is defined 
based on the change in tumor burden (after ruling out irPD). Decreases in tumor burden must 
be assessed relative to baseline measurements (i.e., the SPD of all index lesions at screening). 
 
Overall Response using the irRC: 
The overall response according to irRC is derived from time-point response assessments 
(based on tumor burden) as follows: 
 
irCR: 
Complete disappearance of all lesions (whether measurable or not, and no new 
lesions) confirmed by a repeat, consecutive assessment done no less than 4 weeks 
from the date first documented. 
 
irPR: 
Decrease in tumor burden ≥50%relative to baseline confirmed by a consecutive 
assessment done at least 4 weeks after first documentation 
 
irSD, 
Not meeting criteria for irCR or irPR, in absence of irPD 
 

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irPD 
Increase in tumor burden ≥25% relative to nadir (minimum recorded tumor burden). 
Confirmation by a repeat, consecutive assessment done no less than 4 weeks from the 
date first documented.  
 
 
Reference: 
Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-
Related Response Criteria. Wolchok, JD, Hoos, A, O'Day, S, Weber, JS, Hamid, O, Lebbe  ,י
C, Maio, M, Binder, M, Bohnsack, O, Nichol, G, Humphrey, R, and Hodi, FS, Clin Cancer 
Res 2009;15:7412-7420 
 

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Measurable Response 
         Non-measurable Response 
Overall Response 
 
Index 
and 
new, 
measurable 
lesions 
(tumor burden) 1% 
Non-index lesions 
New, nonmeasurable 
lesions 
Using irRC 
Decrease 100% 
Absent 
absent 
irCR2 
Decrease 100% 
Stable 
Any 
irPR2 
Decrease 100% 
Unequivocal 
progression 
Any 
irPR2 
Decrease > 50% 
Absent/stable 
Any 
irPR2 
Decrease > 50% 
Unequivocal 
progression 
Any 
irPR2 
Decrease< 50% to < 25% 
increase 
Absent/stable 
Any 
irSD 
Decrease< 50% to < 25% 
increase 
Unequivocal 
progression 
Any 
irSD 
Increase > 25% 
Any 
Any 
irPD 
 
 
 
 
1. 
Decreases 
assessed 
relative 
to 
baseline, 
including 
measurable lesions only (>5x5 mm) 
2. 
Assuming response (irCR) and progression (ir PD) are 
confirmed by a second, consecutive assessment done at 
least 4 weeks apart 
 

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12.0 
REFERENCES 
1. 
Robert, C., et al. Anti-programmed-death-receptor-1 treatment with pembrolizumab 
in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort 
of a phase 1 trial. Lancet 384, 1109-1117 (2014). 
2. 
Vivier, E., Tomasello, E., Baratin, M., Walzer, T. & Ugolini, S. Functions of natural 
killer cells. Nature immunology 9, 503-510 (2008). 
3. 
Imai, K., Matsuyama, S., Miyake, S., Suga, K. & Nakachi, K. Natural cytotoxic 
activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up 
study of a general population. Lancet 356, 1795-1799 (2000). 
4. 
da Silva, I.P., et al. Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 
blockade. Cancer immunology research 2, 410-422 (2014). 
5. 
Jayaraman, P., et al. Tim3 binding to galectin-9 stimulates antimicrobial immunity. 
The Journal of experimental medicine 207, 2343-2354 (2010). 
6. 
Chiba, S., et al. Tumor-infiltrating DCs suppress nucleic acid-mediated innate 
immune responses through interactions between the receptor TIM-3 and the alarmin 
HMGB1. Nature immunology 13, 832-842 (2012). 
7. 
Huang Yu-Hwa, Z.C., et al. CEACAM1 regulates TIM-3-mediated tolerance and 
exhaustion. Nature (2014). 
8. 
Moller, M.J., Kammerer, R., Grunert, F. & von Kleist, S. Biliary glycoprotein (BGP) 
expression on T cells and on a natural-killer-cell sub-population. International 
journal of cancer. Journal international du cancer 65, 740-745 (1996). 
9. 
Casado, J.G., et al. Expression of adhesion molecules and ligands for activating and 
costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of 
human melanoma cell lines. Cancer immunology, immunotherapy : CII 58, 1517-
1526 (2009). 
10. 
Hamid, O., et al. Safety and tumor responses with lambrolizumab (anti-PD-1) in 
melanoma. N Engl J Med 369, 134-144 (2013). 
 11. 
Rangachari, M., et al. Bat3 promotes T cell responses and autoimmunity by 
repressing Tim-3-mediated cell death and exhaustion. Nat Med 18, 1394-1400 (2012). 
 

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13.0 
SUPPLEMENTARY TABLES 
 
 
 
Supplementary Table 1: Higher expression of Tim-3 and KIRB1, and lower cytotoxicity, cytokine 
production, proliferation and T-bet expression are associated with higher stages. Values have been 
evaluated by flow cytometry in a total number of 100 patients. Values inside a red square have been 
validated in an independent cohort (n=50). 
1One way analysis of variance (ANOVA) 
2 Unpaired t test 
Supplementary Table 2: Higher expression of Tim-3 and a lower cytotoxicity and cytokine 
production ability are associated with thicker melanomas. Also higher expression of Tim-3 and 
KIRB1, and lower cytotoxicity and T-bet expression are associated with local or distant 
metastases. Values have been evaluated by flow cytometry in a total number of 88 (upper panel) and 
100 patients(lower panel). Values inside a red square have been validated by an independent cohort 
(n=50).  
1One way analysis of variance (ANOVA) 
2