LIMIT Melanoma: (Lysosomal Inhibition + Melanoma ImmunoTherapy) A Phase 1/2 Open Label Trial of Nivolumab and Hydroxychloroquine or Nivolumab/Ipilimumab and Hydroxychloroquine in Patients With Advanced Melanoma
Phase: PHASE1
Conditions: Melanoma
Sponsor: Ravi Amaravadi, MD

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LIMIT Melanoma: (Lysosomal Inhibition + Melanoma ImmunoTherapy) 
A phase 1/2 open label trial of nivolumab and hydroxychloroquine, nivolumab-relatlimab or 
nivolumab/ipilimumab and hydroxychloroquine in patients with advanced Melanoma 
 
Sponsor-Investigator/ 
Principal Investigator: 
Ravi Amaravadi, MD 
University of Pennsylvania Perelman School of Medicine 
Department of Medicine, Division of Hematology Oncology 
3400 Civic Center Blvd, 10th Floor, South Pavilion 
Philadelphia, PA 19104 
Sub-Investigators: 
Lynn Schuchter, MD; Tara Mitchell, MD; Federico Valdivieso;  
Giorgos Karakousis, MD; Alexander Huang, MD, PhD, John Miura, MD;
Phyllis Gimotty, PhD (statistics) 
 
Medical Monitor: 
Noelle Frey, MD, MSCE 
Funding: 
Bristol Myers Squibb (BMS) Investigator Initiated Trial 
Protocol Number: 
IRB:  835033 
UPCC: 01620 
BMS: CA209-7H6 
Investigational Agents: 
Nivolumab: commercial supply 
Ipilimumab: commercial supply 
Nivolumab-relatlimab: commercial supply  
Hydroxychloroquine: commercial supply 
IND Exempt 
ClinicalTrials.gov Identifier: 
NCT04464759 
Initial version: 
Amendment 1 
Amendment 2 
Amendment 3          
Amendment 4                 
12.12.2019 
05.08.2020 
02.28.2021 
01.10.2022 
06.13.2023 
 
 
 

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PRINCIPAL INVESTIGATOR SIGNATURE 
SPONSOR-INVESTIGATOR: 
RAVI AMARAVADI, MD 
STUDY TITLE: 
LIMIT Melanoma: (Lysosomal Inhibition + Melanoma 
ImmunoTherapy)  A phase 1/2 open label trial of nivolumab and 
hydroxychloroquine, nivolumab-relatlimab, or 
nivolumab/ipilimumab and hydroxychloroquine in patients with 
advanced melanoma 
STUDY ID 
PROTOCOL VERSION 
IRB #835033 / UPCC 01620 
6.13.2023 
 
I have read the referenced protocol. I agree to conduct the study in accordance to this protocol, in 
compliance with the Declaration of Helsinki, Good Clinical Practices (GCP), and all applicable regulatory 
requirements and guidelines. 
________________________________________ 
Principal Investigator Name 
_________________________________________ 
Signature 
 
 
________________________________________ 
Affiliation 
_________________________________________ 
Date 
 
 
 
 
 
 

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Abbreviations: 
 
Ab: antibody 
AE: adverse event 
ALT: alanine aminotransferase 
ANC: absolute neutrophil count 
ASCO: American Society of Clinical Oncology 
AST: aspartate aminotransferases 
BAMM: Braf, Autophagy and MEK for Melanoma 
BID: twice daily 
BMS: Bristol-Myers Squibb 
BP: blood pressure 
BSA: body surface area 
CNS: central nervous system 
CR: complete response 
CT: computed tomography 
CTCAE: Common Terminology Criteria for 
Adverse Events 
DLT: dose-limiting toxicity 
DSMC: Data Safety and Monitoring Committee 
ECOG: Eastern Cooperative Oncology Group 
eCRF: electronic case report form 
EKG: electrocardiogram 
ESR: expedited safety report 
FDA: Food and Drug Administration 
FFPE: formalin fixed-paraffin embedded 
HBV: hepatitis B virus 
HCQ: hydroxychloroquine 
HCV: hepatitis C virus 
HUP: Hospital of the University of Pennsylvania 
IB: investigators brochure 
IHC: immunohistochemistry 
IND: investigational new drug 
INR: international normalization ratio 
I-O: immune-oncology 
IRB: institutional review board 
IV: intravenous 
LIMIT: Lysosomal Inhibition + Melanoma 
Immunotherapy 
LLN: lower limit of normal 
LVEF: left ventricular ejection fraction 
MDSC: myeloid derived suppressor cells mg: 
milligrams 
MRI: magnetic resonance imaging 
MTD: maximum tolerated dose 
NCI: National Cancer Institute 
ORR: overall response rate 
OS: overall survival 
PBMC: peripheral blood mononuclear cells 
PD: progressive disease 
PD-1: programmed death-1 
PFS: progression-free survival 
PK: pharmacokinetic 
PMN; polymorphonuclear 
PPT1: palmitoyl-protein thioesterase 1 
PR: partial response 
PT: prothrombin time 
PTT: partial thromboplastin time 
RECIST: The Response Evaluation Criteria in 
Solid Tumors 
SAE: serious adverse event 
SD: stable disease 
SUSAR: Suspected, Unexpected Serious 
Adverse Reaction 
TAM (tumor associated macrophage) 
TIL: tumor infiltrating lymphocytes 
TNF: tumor necrosis factor 
TSH: thyroid stimulating hormone 
ULN: upper limit of normal 
WBC: white blood cell 
 
 
 
  
 

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1.0 
OBJECTIVES ........................................................................................................................ 10 
1.1 
PRIMARY OBJECTIVE ............................................................................................................... 10 
1.2 
SECONDARY OBJECTIVES ........................................................................................................ 10 
1.3 
CORRELATIVE OBJECTIVES ..................................................................................................... 10 
 
2.0 
BACKGROUND AND RATIONALE ...................................................................................... 11 
2.1 
CLINICAL UNMET NEED ........................................................................................................... 11 
2.2 
CLINICAL DEVELOPMENT OF PD-1 INHIBITORS IN MELANOMA. .................................................... 11 
2.3 
AUTOPHAGY IN CANCER .......................................................................................................... 12 
2.4 
HYDROXYCHLOROQUINE .......................................................................................................... 11 
2.5 
POSITIVE PHASE II STUDIES INVOLVING HCQ IN MELANOMA AND OTHER CANCERS .................... 122 
2.6 
PRECLINICAL DATA INDICATES ENHANCED EFFICACY WITH ANTI-PD1 + HCQ COMBINATION 
THERAPY. ................................................................................ ERROR! BOOKMARK NOT DEFINED. 
2.7 
HCQ, LYS05 AND DQ661 ARE TARGETED THERAPIES. .............................................................. 12 
2.8 
LYSOSOMAL INHIBITION PROMOTES M2 (IMMUNOSUPPRESSIVE) TO M1 (ANTITUMOR) PHENOTYPE 
SWITCH IN TUMOR ASSOCIATED MACROPHAGES. ........................ ERROR! BOOKMARK NOT DEFINED. 
2.9 
EXTENSIVE CLINICAL CHARACTERIZATION OF ANTI-PD1 AB IMMUNE EFFECTS AT PENN. ............. 13 
2.10 BLOOD- AND TISSUE-BASED PHARMACODYNAMIC BIOMARKERS ENABLE THE ASSESSMENT OF 
THERAPEUTIC MODULATION OF AUTOPHAGY. ........................................................................... 13 
2.11 RATIONALE FOR CORRELATIVE STUDIES ................................................................................... 14 
 
3.0 
PATIENT SELECTION .......................................................................................................... 15 
3.1 
INCLUSION CRITERIA ............................................................................................................... 15 
3.2 
EXCLUSION CRITERIA: ............................................................................................................. 16 
 
4.0 
TREATMENT PLAN .............................................................................................................. 18 
 
5.0 
DETAILS OF STUDY TREATMENT ..................................................................................... 20 
5.1 
HYDROXYCHLOROQUINE .......................................................................................................... 20 
5.2 
NIVOLUMAB ............................................................................................................................ 20 
5.3 
IPILIMUMAB ............................................................................................................................. 21 
5.4 
CONCOMITANT MEDICATION, DRUG-DRUG INTERACTIONS AND PROCEDURES ............................ 23 
5.5 
DURATION OF PROTOCOL TREATMENT AND FOLLOW-UP. .......................................................... 24 
 
6.0 
DOSE LIMITING TOXICITY AND DOSE ESCALATION RULES .......................................... 24 
6.1 
DEFINITION OF DOSE LIMITING TOXICITY (DLT) ......................................................................... 24 
6.2 
DOSE ESCALATION RULES ...................................................................................................... 25 
6.3 
DOSE LEVELS ......................................................................................................................... 25 
6.4 
SINGLE ARM PHASE 2 TRIAL WITH 2 COHORTS .......................................................................... 26 
 
7.0 
TOXICITY CRITERIA, MONITORING, DOSE DELAYS AND MODIFICATIONS .................. 26 
7.1 
TOXICITY CRITERIA.................................................................................................................. 26 
7.2 
DOSE DELAYS ........................................................................................................................ 26 
7.3 
OCULAR TOXICITY ................................................................................................................... 26 
7.4 
NIVOLUMAB SIDE EFFECTS OVERVIEW ..................................................................................... 26 
7.5 
TREATMENT OF NIVOLUMAB RELATED INFUSION REACTIONS .................................................... 27 
7.6 
IPILIMUMAB SIDE EFFECTS OVERVIEW ...................................................................................... 28 
7.7 
IPILIMUMAB AND NIVOLUMAB ................................................................................................... 28 
7.8 
MONITORING AND DOSE DELAY CRITERIA ................................................................................ 31 
7.9 
MANAGEMENT GUIDELINES FOR IMMUNE RELATED ADVERSE EVENTS ......................................... 31 
7.10 HYDROXYCHLOROQUINE DOSE REDUCTION .............................................................................. 36 
7.11 DOSE REDUCTIONS FOR TOXICITIES THAT OVERLAP NIVOLUMAB, IPILIMUMAB AND HCQ .............. 37 
 

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8.0 
CORRELATIVE STUDIES ..................................................................................................... 37 
8.1 
ESTIMATED NUMBER OF SUBJECTS ........................................................................................... 37 
8.2 
SERIAL TUMOR BIOPSIES AND ANALYSIS .................................................................................. 37 
8.3 
IMMUNOPHENOTYPING ON SERIALLY COLLECTED BLOOD AND TUMOR TISSUE ............................. 38 
8.4 
TRACKING TUMOR SPECIFIC T CELLS USING COMBINATORIAL TETRAMER .................................... 38 
8.5 
IMMUNOHISTOCHEMISTRY FOR MARKERS OF TUMOR IMMUNITY IN SERIAL BIOPSIES ..................... 38 
8.6 
NANOSTRING ON SERIALLY COLLECTED TUMOR TISSUE ............................................................. 39 
8.7 
FFPE ARCHIVAL TUMOR TISSUE FOR IMMUNOHISTOCHEMISTRY ................................................. 39 
 
9.0 
STUDY CALENDAR (TABLE 6) ........................................................................................... 40 
 
10.0 MEASUREMENT OF EFFECT .............................................................................................. 44 
10.1 DEFINITIONS ........................................................................................................................... 44 
10.2 DISEASE PARAMETERS ............................................................................................................ 44 
10.3 METHODS FOR EVALUATION OF MEASURABLE DISEASE ............................................................ 45 
10.4 RESPONSE CRITERIA ............................................................................................................... 45 
10.5 DURATION OF RESPONSE ........................................................................................................ 47 
10.6 PROGRESSION-FREE SURVIVAL ............................................................................................... 47 
10.7 OFF TREATMENT/OFF STUDY ................................................................................................... 47 
 
11.0 ADVERSE EVENTS AND REPORTING ............................................................................... 47 
11.1 ADVERSE EVENTS ................................................................................................................... 47 
11.2 RECORDING OF ADVERSE EVENTS ........................................................................................... 48 
11.3 ASSESSMENT OF ADVERSE EVENTS ......................................................................................... 52 
11.4 UPENN ABRAMSON CANCER CENTER (ACC)’S DATA AND SAFETY MONITORING COMMITTEE 
(DSMC) NOTIFICATION BY ALL SITES ....................................................................................... 53 
11.5 UPENN IRB NOTIFICATION BY INVESTIGATOR-SPONSOR .......................................................... 54 
11.6 MEDICAL MONITORING ............................................................................................................ 56 
11.7 STUDY MONITORING PLAN ....................................................................................................... 56 
11.8 AUDITING AND INSPECTING ........................................................................................................ 56 
 
12.0 STATISTICAL CONSIDERATIONS ...................................................................................... 56 
12.1 PHASE 1 DESIGN ..................................................................................................................... 56 
12.2 SAMPLE SIZE CALCULATION FOR PHASE 2 ................................................................................ 57 
12.3 ANALYSIS OF SECONDARY ENDPOINTS..................................................................................... 57 
REFERENCES ................................................................................................................................ 58 
APPENDIX A ................................................................................................................................... 62 
APPENDIX B ................................................................................................................................... 63 
 
 
 

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STUDY SUMMARY 
Title 
LIMIT Melanoma: (Lysosomal Inhibition + Melanoma 
ImmunoTherapy)  A phase 1/2 open label trial of nivolumab and 
hydroxychloroquine, nivolumab/ipilimumab and 
hydroxychloroquine, or nivolumab-relatlimab and 
hydroxychloroquine in patients with advanced melanoma 
Protocol Number 
UPCC: 01620; IRB #835033; BMS: CA209-7H6 
Phase 
Phase 1/2 
Methodology 
Non-randomized open label 4-part phase 1/2 
 
Part 1: Phase 1a dose escalation study with nivolumab + HCQ.  After 
the maximum tolerated dose of HCQ has been identified, patients 
will be recruited for one of the following: 
Part 2: Phase 2 expansion cohort of nivolumab + HCQ; OR 
Part 3: Phase 1 dose escalation with nivolumab, ipilimumab and 
HCQ (3-drug combination) 
Part 4: Phase 2 nivolumab-relatlimab + HCQ 
Study Duration 
5 years 
Study Center(s) 
Single Institution  
Objectives 
PRIMARY OBJECTIVES: 
 
Phase 1a nivolumab + HCQ:  To evaluate the safety and tolerability 
of HCQ administered in combination with nivolumab in subjects with 
advanced/metastatic melanoma, and to select the maximum 
tolerated dose (MTD) of HCQ for further evaluation 
 
Phase 2 (expansion cohorts for nivolumab + HCQ):  To assess 
the objective response rate (ORR) as measured by RECIST v1.1. in 
subjects with advanced melanoma that are treatment-refractory to 
anti-PD-1 antibody therapy (Cohort 2a) or treatment naïve to anti-
PD-1 antibody-based therapy (Cohort 2b) 
 
Phase 1b Ipilimumab + Nivolumab + HCQ (3-drug combination):  
To evaluate the safety and tolerability of HCQ administered in 
combination with nivolumab and ipilimumab followed by maintenance 
nivolumab in subjects with advanced/metastatic melanoma, and to 
select the maximum tolerated dose (MTD) of HCQ for further 
evaluation 
 
Phase 2c Nivolumab- relatlimab + HCQ: To assess the objective 
response rate (ORR) as measured by RECIST v1.1. in subjects with 
advanced melanoma that are treatment-refractory to anti-PD-1 
antibody therapy 
 

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STUDY SUMMARY 
SECONDARY OBJECTIVES: 
To estimate other measures of anti-tumor activity, including 
progression-free survival (PFS), and duration of response, following 
combination therapy, 1-year survival rate 
 
CORRELATIVE OBJECTIVES: 
1) To measure changes in tumor microenvironment by IHC in serial 
tumor biopsies including MART1 (melanoma), CD8+ (cytotoxic T 
cells), F4/80 (TAM), Ly6G/C (MDSC). Using multiplexed IHC we will 
be able to determine if the addition of HCQ reduces PMN-MDSC, or 
converts M2 to M1 TAMs, and results in increased infiltration of T 
cells into the tumor 
2) To evaluate the association between baseline tumor tissue 
expression of beclin1, LC3, p62, HLTF, and ALDH1A1, PPT1 and 
response to combination therapy 
3) To characterize the pharmacodynamic impact of combination 
therapy on markers of immune modulation in the hypoxic tumor 
microenvironment, including tumor infiltrating lymphocytes, M2 to M1 
macrophages, MDSC, and tumor specific T cells in the periphery 
4) To determine if exosomal PDL1 can predict response to 
nivolumab + HCQ or nivolumab-relatlimab + HCQ  
Number of Subjects 
Original Planned Sample Size  
Phase 1a nivolumab and HCQ Dose Escalation:  6-12 patients 
 
Phase 2 nivolumab and HCQ 
-- Cohort 2a (anti-PD1 Ab refractory): 27 patients (closed early)  
-- Cohort 2b (anti-PD1 Ab naive): 43 patients (closed early)  
 
 
Phase 1b nivolumab, ipilimumab and HCQ (3-drug combination) 
Dose Escalation:  6-12 patients 
 
Total: up to 94 patients 
2023 Amendment Sample Size  
Phase 1a: 7 
Phase 1b: 6-12 
Phase 2a-2b: 9 
Phase 2c Phase 2 nivolumab-relatlimab+HCQ 
 Cohort 2c anti-PD-1 Ab refractory): 35 patients  
 
Total Sample Size: Up to 63 
 
 
 

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STUDY SUMMARY 
 
Diagnosis and Main 
Inclusion/Exclusion 
Criteria 
1. Histologic or cytologic evidence of melanoma Stage III 
unresectable or Stage IV, any genotype, and any PD-L1 IHC 
status 
2. Phase 1a nivolumab + HCQ: any prior treatment, or treatment 
naïve Phase 2 nivolumab + HCQ: 
-- Cohort 2a: prior immunotherapy in the adjuvant or metastatic 
setting is required 
-- Cohort 2b: patients must be anti-PD-1 Ab-naïve 
Phase 2 nivolumab-relatlimab + HCQ: 
Cohort 2c: prior anti-PD-1 antibody  in the adjuvant or metastatic 
setting is required 
Phase 1b nivolumab + ipilimumab + HCQ: patients must be anti-
PD-1 refractory 
3. ECOG performance status of 0-1 
4. At least one measurable site of disease by RECIST 1.1 criteria 
that has not been previously irradiated 
5. Fresh or archived primary or metastatic tissue available for 
submission for correlative analyses 
6. Patients with history of treated brain metastases are eligible if off 
systemic corticosteroids for at least 1 week 
7. Patients must have normal organ function acceptable for 
combination therapy 
Study Product, Dose, 
Route, Regimen 
All study products obtained commercially 
Phase 1a nivolumab + hydroxychloroquine 
Hydroxychloroquine 400-600 mg po q12 h and nivolumab 480 mg IV 
every 4 weeks 
 
Phase 2 nivolumab + hydroxychloroquine 
Hydroxychloroquine 400-600 mg po q12 h (MTD from Phase I) and 
nivolumab 480 mg IV every 4 weeks 
 
Phase 2c: nivolumab-relatlimab + hydroxychloroquine  
Hydroxychloroquine 600 bid mg po and nivolumab 480 mg + 
relatlimab 160 mg IV every 4 weeks 
  
Phase 1b nivolumab + ipilimumab + hydroxychloroquine (3-drug 
combination): 
Hydroxychloroquine 400-600 mg po q12 h + nivolumab 1mg/kg IV 
plus ipilimumab 3 mg/kg IV every 3 weeks x4 cycles 
then 6 weeks after the last dose of ipi/nivo begin maintenance 
nivolumab 480 mg IV every 4 weeks 
Duration of 
administration 
Until progression or up to 24 months of therapy if partial or complete 
response. 

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STUDY SUMMARY 
Study design  
Phase 1 (both 2- and 3-drug combinations) include a 3+3 dose 
escalation to determine MTD. Phase 2 cohorts each have a 2-stage 
design 
Duration of trial 
Approximately 5 years 
 
Follow-up: 
o All subjects will be evaluated per study calendar for 100 days 
after discontinuation of study treatment. 
o All subjects will be followed for a minimum of one year from the 
start of treatment,unless disease progression is reported prior 
to one year. 
 
 
1.0 OBJECTIVES 
1.1 Primary Objective 
Phase 1a nivolumab and hydroxychloroquine (HCQ):  To determine the maximum tolerated dose (MTD) 
and preliminary safety of HCQ when administered in conjunction with nivolumab in patients with advanced 
melanoma. 
Phase 1b nivolumab and ipilimumab and HCQ (following Phase I nivolumab and HCQ): To determine the 
maximum tolerated dose (MTD) and preliminary safety of hydroxychloroquine (HCQ) when administered 
in conjunction with nivolumab and ipilimumab in patients with advanced melanoma. 
Phase 2 (Cohorts 2a , 2b, 2c following Phase 1a nivolumab and HCQ): To assess the clinical efficacy  as 
measured by response rate. 
1.2 Secondary Objectives 
1.2.1 To estimate the toxicity rates 
1.2.2 To estimate progression-free survival 
1.2.3 To estimate 1 year survival rates 
1.2.4 Duration of therapy  
1.3 Correlative Objectives 
1.3.1 To measure changes in tumor microenvironment by IHC in serial tumor biopsies including MART1 
(melanoma), CD8+ (cytotoxic T cells), F4/80 (TAM), Ly6G/C (MDSC). Using multiplexed IHC we 
will be able to determine if the addition of HCQ reduces PMN-MDSC, or converts M2 to M1 TAMs, 
and results in increased infiltration of T cells into the tumor. 
1.3.2 To evaluate the association between baseline tumor tissue expression of beclin1, LC3, p62, HLTF, 
and ALDH1A1, PPT1 and clinical outcome on combination therapy. 
1.3.3 To characterize the pharmacodynamic impact of combination therapy on markers of immune 
modulation in the hypoxic tumor microenvironment, including tumor infiltrating lymphocytes, M2 to 
M1 macrophages, MDSC, and tumor specific T cells in the periphery. 

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1.3.4 To determine if exosomal PDL1 can predict response to nivolumab + HCQ or nivolumab-relatlimab 
+ HCQ 
 
2.0 BACKGROUND AND RATIONALE 
2.1 Clinical Unmet Need 
Novel combination approaches are needed to improve clinical response rates with anti-PD1 
therapy. Anti-PD1 immunotherapies have achieved unprecedented clinical benefit for melanoma with 
response rates of 33-40% as single agents 1.  Efforts to enhance this response rate have focused on 
combination with other immune checkpoints but besides the anti-PD1 Ab and anti-CTLA4 antibody strategy 
(58% response rate), which is associated with much higher rates of serious toxicity 2, no clearly superior 
combination has emerged.  As such, novel combinatorial treatment strategies that are tolerable and 
increase the response rate represent a major unmet need.  Our group has focused on targeting 
autophagy in cancer with the antimalarial compound hydroxychloroquine (HCQ) 3. Previously we have 
found that autophagy is increased in melanoma 4. We then found that BRAF inhibition induces 
cytoprotective autophagy, and HCQ can augment BRAF inhibitor therapy in animal studies 5. More recently 
we have found that MAPK reactivation, which is the more commonly accepted mechanism of resistance to 
targeted therapy in melanoma drives autophagy induction (Ojha Cancer Discovery 2019). Therefore in 
targeted therapy for melanoma autophagy seems to be a major common resistance mechanism. These 
findings have led to a clinical trial of dabrafenib + trametinib + HCQ in BRAF mutant melanoma that has 
found striking response rates (see below for details). In parallel, our focus on targeting autophagy in cancer 
has led to the new discovery of the lysosomal enzyme PPT1 as an attractive new target in melanoma and 
other cancers 6. New data indicates the molecular target of the lysosomal inhibitor HCQ is PPT1 (Rebecca 
et al Cancer Discovery 2019), and targeting PPT1 in the tumor microenvironment concurrently impairs 
tumor cell growth and results in M2 to M1 macrophage phenotype switching that enhances tumor immunity 
7. Based on these findings we performed a preclinical study in the B16 mouse model and found that HCQ 
significantly augmented anti-PD1 Ab in this model as well as a genetically engineered mouse model of 
BRAF mutant melanoma with an intact immune system (see below). These findings provide the rationale 
for pursuing the combination of PD1 Ab and HCQ. If this combination appears safe and effective in single 
arms study this paves the way for a 3 drug combination, or a randomized study to be conducted through 
ECOG. 
 
2.2 Clinical development of PD-1 inhibitors in melanoma. 
PD-1 is a negative regulator of T-cell activity that limits the activity of T cells at a variety of stages of the 
immune response when it interacts with its two ligands PD-L1 and PD-L2 8. PD-1 is primarily believed to 
inhibit effector T-cell activity in the effector phase within tissues. This pathway is likely important in the 
tumor microenvironment where PD-L1 expressed by tumors interacts with PD-1 on T cells to suppress T-
cell effector function by limiting T-cell receptor localization to the immunologic synapse at the site of target 
engagement. The FDA approved anti-PD-1 antibodies pembrolizumab and nivolumab have robust clinical 
activity. In multiple clinical trials in patients with advanced melanoma nivolumab 2 mg/kg given every 2 
weeks produced a response rate of 31%-44% depending on the median length of followup 1, 2. Updated 
results from Keynote-006 which randomized patients with advanced melanoma to 10 mg/kg 
pembrolizumab every 2 versus 3 weeks versus ipilimumab 3 mg/kg every 3 weeks showed a similar 
response rate in 2 versus 3 week dosing schedules of 36-37% 9. Longer term followup shows that 
depending on the makeup of the patient population response rates with 3-4 year followup shows that 
pembrolizumab can produce up to a 42% response rate (Long et al. ASCO abstract 2018).  The sum of 
these data lead to the FDA approval of both pembrolizumab and nivolumab for the treatment of metastatic 
melanoma. Nivolumab given every 4 weeks at 480 mg showed equivalent PK properties to nivolumab 240 
mg given every 2 weeks, and due to the longer schedule has become the de facto most used PD-1 Antibody 
regimen 10. PD-L1 IHC is not required for PD1 inhibitor therapy in melanoma. The combination of 
ipilimumab and nivolumab produces a response rate of 58% but there has not been a significant difference 

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in overall survival demonstrated when comparing ipilimumab and nivolumab compared to nivolumab alone 
11. In exchange for these differences in efficacy ipilimumab and nivolumab produced a 48% grade 3 toxicity 
and 11% grade 4 toxicity in patients compared to nivolumab which produced a 17% grade 3, and 5% grade 
4 toxicity rate. 
 
2.3 Autophagy in Cancer 
Autophagy is an intrinsic cellular resistance mechanism induced by therapeutic stress from anti-
cancer agents, and serves a key role in immune resistance within the hypoxic tumor 
microenvironment. Autophagy is an intracellular mechanism in which toxic byproducts of cellular 
metabolism are engulfed by autophagic vesicles (AV) and disposed via lysosomal degradation 12. Thus, 
autophagy prevents the accumulation of toxic products and maintains metabolic homeostasis through the 
recycling of cellular components. This highly regulated process is an emerging hallmark of cancer, in which 
autophagy supports the survival of established malignancy under challenging metabolic conditions, such 
as nutrient/growth factor deprivation or hypoxia.  As such, upregulation of autophagy has been identified 
as an intrinsic tumor resistance mechanism to therapeutic stress induced by systemic anti-cancer 
therapies, including both traditional cytotoxic and targeted agents. For example, the induction of 
cytoprotective autophagy has been demonstrated in response to alkylating agents, as well as to BRAF 
inhibition in BRAF-mutant melanoma.  These findings have laid the mechanistic rationale for several clinical 
investigations of combination chemotherapy or targeted therapy with inhibitors of cancer cell autophagy 
that have already been conducted by our group. 3 
 
Importantly, autophagy is highly upregulated in the hypoxic tumor microenvironment, and serves a key role 
in the cancer cell- tumor microenvironment interaction 13. In this context, recent preclinical evidence 
demonstrates that autophagy helps define the anti-tumor immune response and immune effector cell 
resistance. For example, activation of autophagy in the hypoxic tumor microenvironment leads to the 
upregulation of phosphorylated STAT3, resulting in impaired cytotoxic T cell-mediated tumor lysis 14.  In 
addition, autophagy contributes to increased degradation of natural killer cell-derived granzyme B in 
hypoxic tumor cells.  A very recent report demonstrates that knockout of the essential autophagy gene 
Beclin leads to augmented immune infiltration into the tumor microenvironment 15. However, our own 
preliminary data demonstrates that Atg7 knockout in the tumor cell alone does not augment PD1 Ab 
efficacy (see below), whereas targeting the lysosome with HCQ does augment PD1 Ab efficacy (see 
below). This data likely reflects the fact that ATG7 is required for secretion programs, which may be critical 
for the efficacy of anti-PD1 therapy. Targeting the lysosome is the only clinically feasible way of inhibiting 
autophagy to enhance PD1 Ab efficacy. 
 
2.4 Hydroxychloroquine 
Hydroxychloroquine is a well -tolerated lysosomal autophagy inhibitor used in humans for > 50 
years. Hydroxychloroquine (HCQ) is a commercially available oral medication that is commonly used for 
the treatment of rheumatoid arthritis at doses of 400 mg daily.  Chloroquine derivatives, such as HCQ, are 
known to modulate autophagy by blocking the delivery of autophagic contents to the lysosome for 
degradation.  Thus, by blocking the final step of autophagy, HCQ can lead to the accumulation of 
cytoplasmic AVs.  Several early phase clinical trials were published by our group involving HCQ as a 
mechanism for autophagy inhibition in combination with a variety of targeted agents in cancer patients.  In 
these studies, HCQ was evaluated in combination with vorinostat, bortezomib, temozolomide/radiation 
therapy, and temsirolimus in a variety of solid tumors 16-20.  In general, these studies reported an overall 
acceptable toxicity profile with these combination therapies, with primary toxicities of low-grade nausea, 
diarrhea, and myelosuppression.  The recommended phase II dose of HCQ in combination with targeted 
agents has ranged from 600 mg daily to 1200 mg daily in divided doses. 
 

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2.5 Phase II studies involving HCQ in melanoma and other cancers 
Indeed, more recent phase II studies by our group involving HCQ have yielded striking results. A phase I/II 
study of FOLFOX + Avastin + HCQ has produced a high response rate (70% ORR, 11% CR) with no 
additional observed toxicity (NCT01206530; manuscript under revision).  Similarly, in BRAF mutant 
melanoma, the phase I/II trial of dabrafenib, trametinib, and HCQ has produced a 85% response rate and 
41% complete response rate (NCT02257424;Mehnert et al Clinical Cancer Research 2022). Most 
excitingly, a neoadjuvant study of gemcitabine + abraxane +/- HCQ found that the arm that received HCQ 
had a significantly better pathological response rate, reduced lymph node metastases, and reduction in 
serum CA19-9 (Zeh et al. Clinical Cancer Research 2021).. Finally, in clear cell renal cell carcinoma, a 
phase I/II combination of HCQ with everolimus led to an increased PFS when compared to the historical 
control of everolimus alone (NCT01510119; Hass Clinical Cancer Research 2019).  In most of these trials 
the addition of high dose HCQ (600 mg bid) did not produce significant adverse events. Additional clinical 
trials that demonstrated activity of HCQ regimens were in 3rd line colon cancer 21, and multiple myeloma. 
 
2.6 HCQ, Lys05 and DQ661 are targeted therapies 
The only clinically available autophagy inhibitors are the CQ derivatives. We have also generated a series 
of more potent dimeric aminoquinolines, Lys05 and DQ661 6, 22.  We recently showed that DQ661 binds 
and inhibits the lysosome enzyme palmitoyl-protein thioesterase 1 (PPT1), which regulates intracellular 
trafficking of receptors and secreted proteins through palmitoylation. Many of the autophagy, mTOR, 
metabolic enzyme, solute carriers, and interferon signaling proteins are regulated by palmitoylation. We 
now have data that HCQ and Lys05 also bind to and inhibit this enzyme (Rebecca et al Cancer Discovery 
2019). Therefore HCQ and more potent derivatives can be considered targeted therapies and not simply 
weak bases. The newer derivatives are being developed through a biotech company, but are still not 
clinically available. Therefore this proposal will utilize HCQ as the proof-of-principle PPT1 inhibitor 
that is ready for immediate clinical translation. 
 
 
 
 
2.7 Rationale for amendment 4. 
  
The increased progression-free survival observed with relatlimab and nivolumab (nivo-rela) compared to 
nivolumab has reinvigorated the melanoma community. With less toxicity than nivolumab and ipilimumab, 
the nivo-rela combination is very promising, but efficacy in second-line PD-1 antibody refractory patients 
remains poor with a 12% response rate in a heavily pre-treated population24. There is substantial evidence 
that targeting autophagy can enhance the efficacy of immune checkpoint inhibition through multiple 
Fig 2.  HCQ treatment simultaneously 
impairs tumor cell growth while 
inducing M2-M1 macrophage 
phenotype switching that enables T-cell 
mediated killing.  TAM: tumor associated 
macrophage, MDSC: myeloid derived 
suppressor cell; DC: dendritic cell 

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mechanisms (detailed below). There may be an especially important interaction between 
autophagy/lysosome and the LAG3 receptor. The proposed amendment to the LIMIT melanoma trial will 
establish the preliminary safety and antitumor activity of nivo-rela with the well-tolerated autophagy inhibitor 
HCQ.   
 
2.8 Rationale for combined immune checkpoint and autophagy inhibition.  
 
Recently autophagy inhibition has been identified by 6 separate groups as a strategy to improve the efficacy 
of immune checkpoint inhibition 25-30. A number of mechanisms have been proposed to explain these 
observations. These include the fact that MHC Class I molecules expressed by tumor cells are specific 
autophagy cargo. Autophagy inhibition especially using lysosomal inhibitors enhances surface expression 
and MHC Class I-mediated antigen presentation 25. Autophagy inhibition with chloroquine derivatives also 
induces macrophage repolarization from an M2 to M1 phenotype enabling CD8+ T cell-mediated killing. 
Key to this mechanism is that STING is a specific lysosomal substrate, so lysosomal inhibition increases 
STING levels 26. 
 
2.9 Rationale for blocking the lysosome to enhance LAG3 receptor engagement.  
 
It has been long known that LAG3 is lysosomal substrate. Much of the LAG3 in cells is in the endocytic-
lysosomal compartment 31. LAG-3 trafficking from lysosomal compartments to the cell surface is dependent 
on the cytoplasmic domain through protein kinase C signaling in activated T cells 32. There is some 
evidence that endo/lysosomal recycling following ligand interactions are required to maintain 
immunosuppressive signaling in activated T cells 33.  Lysosomal inhibition likely disrupts physiological 
receptor recycling leading to enhanced but dysfunctional cell surface expression of LAG3. It remains 
unknown if the roughly 10 µM concentrations of HCQ achieved in the blood of cancer patients is sufficient 
to enhance cell surface expression of LAG3, but it is enough to enhance MHC Class I expression in tumor 
cells, which may ultimately be the mechanism by which HCQ can augment nivo-rela.  Therefore, there is 
sufficient rationale and an opportunity to safely test the combination of HCQ with niv-rela in advanced 
melanoma patients who have progressed on PD-1 based therapy.  
 
 
2.10 Extensive clinical characterization of anti-PD1 Ab immune effects at Penn. 
Our group has worked closely with Drs. Alex Huang and John Wherry at Penn to understand the effects of 
pembrolizumab both in the tumor microenvironment and in peripheral blood samples.  Using advanced 
immunophenotyping techniques, we have performed comprehensive immune profiling of tissue and 
peripheral blood, with clinical correlation 34. 
 
 2.11 Blood- and Tissue-based Pharmacodynamic Biomarkers Enable the Assessment of 
Therapeutic Modulation of Autophagy. 
The successful modulation of autophagy can be evaluated via a variety of peripheral blood and tissue-
based pharmacodynamic biomarkers.  In the aforementioned initial published clinical trials, the on-target 
effect of HCQ in blocking the final step of autophagy was demonstrated by the accumulation of AVs in 
peripheral blood mononuclear cells and tumor cells, as determined by electron microscopy.  Further, as 
the ubiquitin-like protein LC3 is integrated into the autophagosome lipid bilayer during normal cellular 
autophagy, changes in LC3 expression by immunoblotting or immunohistochemistry can indicate 
successful modulation of the autophagy program.  Finally, recent evidence has identified candidate 
secreted proteins that are associated with tumor autophagy levels in melanoma 35.  Thus, quantitative 
measurement of these secreted proteins may serve as a pharmacodynamic marker for changes in 
intracellular, tumor-based autophagic activity. 
 
In addition, our group has recently evaluated potential predictors of sensitivity to chloroquine derivatives in 
cancer.  We have performed differential gene expression profiles in both HCQ-sensitive and HCQ-resistant 
cancer cells.  We have determined that combined expression patterns of aldehyde dehydrogenase1A1 

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(ALDH1A1) and helicase like transcription factor (HLTF) identify and differentiate HCQ-sensitive and HCQ-
resistant cancer cells.  Furthermore, using mechanistic studies, we have shown that ALDH1A1 
overexpression enhances entry and cytotoxicity of lysosomal autophagy inhibitors (i.e. enhanced drug 
influx), and that HLTF expression allows for repair of DNA damage caused by lysosomal autophagy 
inhibitor-induced reactive oxygen species, therefore leading to HCQ resistance.  As a result, the 
ALDH1A1HIGH/HLTFLOW 
expression 
profile 
is 
associated 
with 
HCQ-sensitivity, 
whereas 
ALDH1A1LOW/HLTFHIGH is associated with HCQ-resistance.  Importantly, tumor RNA sequencing data from 
>700 patients with advanced solid tumors from the TCGA was queried, and notably RCC and melanoma 
were tumor types with the most prevalent HCQ-sensitive gene expression profiles 36. 
 
Finally we have recently identified the molecular target of HCQ as palmitoyl-protein thioesterase 1 (PPT1). 
This is a depalmitoyalase in the lysosomal that regulates the localization of vacuolar type ATPase 6. V-
ATPase mislocalization following PPT1 inhibition results in lysosomal deacidification and autophagy 
inhibition. High concentrations of HCQ are required for PPT1 inhibition. We have developed a PPT1 IHC 
assay and this will be used to determine if PPT1 expression is associated with PFS in patients treated with 
nivolumab and HCQ. 
 
2.12 Rationale for correlative studies 
To test the hypothesis that HCQ could enhance the therapeutic efficacy of nivolumab by inhibiting the 
lysosome, this clinical trial will incorporate several correlative studies.  First, we will characterize the PK 
properties of HCQ when administered to patients receiving nivolumab. We anticipate that one third of the 
phase II portion of this trial will include biopsies before and after nivolumab + HCQ,nivolumab and 
ipilimumab + HCQ, or nivolumab-relatlimab + HCQ. This will allow pathological analysis including electron 
microscopy by which we can assess the degree of autophagy. This is feasible since the inhibition of the 
last step of autophagy by HCQ should cause an accumulation of autophagic vesicles that can be detected 
by electron microscopy and immunoblotting. We will also perform immunophenotyping on tumors to 
characterize the makeup of their immune infiltrates before and on therapy. Third, through serial blood 
draws, we can investigate non-invasive markers by which autophagic dynamics may be detected. 
 
Tumor based assays: 
The correlative studies will generate preliminary data to address the following questions regarding immune 
checkpoint inhibitor  and HCQ treatment: 
1) 
Is autophagy induced (immune checkpoint inhibitor and blocked (HCQ) effectively? 
2) 
Are there viable biomarkers by which autophagy dynamics can be assessed noninvasively? 
3) 
What are the immune consequences of combined anit PD1 Ab and autophagy blockade? 
Specifically what happens to the M2 and M1 macrophage populations, CD8+ and Treg 
populations and NK cell populations during treatment? 
4) 
What are therapy associated changes in gene expression during response and at progression? 
To answer these questions we will use the following technology: 
Immunophenotyping of one core: Dr. Alexander Huang in John Wherry’s lab at University of Pennsylvania 
has pioneered immunophenotyping of peripheral blood (measuring re-invigoration of exhausted phenotype 
CD8 T cells) in melanoma patients treated with anti-PD1 antibody pembrolizumab 34 . It is currently 
unknown whether a similar immunological response can be identified in patients on anitPD1 Ab and 
autophagy targeted therapies.  We will collect CD45 cells infiltrating the tumor and use multi-parameter 
flow cytometry to characterize immune subpopulations. 
Blood based assays 

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Plasma based biomarkers of autophagy modulation. Recent published data suggests that autophagy is 
not only involved in degradation of dysfunctional proteins but also plays a role in the maturation and 
secretion of functional proteins as well. By profiling the tumor secretome in cell culture and mouse 
xenograft model, we have identified a number of candidate proteins. These have been validated in 
human samples, and in vitro data demonstrate elevations in IL-8, Interleukin-1 Beta, LIF, DKK3, and 
FAM3C in high autophagy states compared to low autophagy states 35. We therefore propose to analyze 
serum concentrations of these proteins in the presence and absence of HCQ and compare them to the 
gold standard of electron microscopy. We can then determine whether these proteins would be useful as 
an non-invasive biomarker of autophagy dynamics. 
 
Plasma exosomal PDL1 can predict response to anti-PD1 antibody. Exosomes are lipid-encapsulated small 
vesicles secreted by cells to the extracellular milieu. Recently Wei Guo’s lab at PENN found that metastatic 
melanoma cells secrete high levels of exosomes that carry a high level of PD-L1 proteins, which potently 
suppresses the function of CD8+ T cells in circulation and facilitates tumor growth 37. In patients with 
metastatic melanoma, the pre-treatment level of circulating exosomal PD-L1 stratifies responders from 
non-responders to anti-PD1 therapy. We also found that the level of exosomal PD-L1 changes during the 
course of anti-PD1 therapy. The magnitude of the early on-treatment changes in exosomal PD-L1 follows 
T cell re-invigoration, and is associated with patient response to therapy. Our research unveils a novel 
mechanism by which tumor cells suppress the immune system, and provides a rationale for a personalized 
approach to monitor patient response to anti-PD1 therapies. 
3.0 PATIENT SELECTION 
3.1 Inclusion Criteria 
3.1.1 Histological or cytological evidence of melanoma, unresectable Stage III or Stage IV, any genotype, 
and any PD-L1 IHC status 
3.1.2 Phase 1a nivolumab + HCQ: any prior treatment, or treatment naïve 
      Phase 2 nivolumab + HCQ: 
            Cohort 2a: prior immunotherapy in the adjuvant or metastatic setting is required 
Cohort 2b: patients must be anti-PD-1 Ab-naïve, but may have received any prior other 
therapy 
Phase 2c (Nivolumab+relatlimab + HCQ): prior PD-1 based immunotherapy in the adjuvant or 
metastatic setting is required; prior anti LAG3 antibody is not allowed.  
      Phase 1b nivolumab + ipilimumab + HCQ: patients must be anti-PD-1 refractory 
3.1.3 ECOG performance status of 0-1 
3.1.4 Age 18 years of age or older 
3.1.5 Patients must have at least one measurable site of disease by RECIST 1.1 criteria that has not 
been previously irradiated. If there is only one RECIST 1.1 measurable lesion, serial research 
biopsies will not be pursued, but the patient is eligible 
3.1.6 Patients must have fresh or archived primary or metastatic tissue available for submission for 
correlative analyses 
3.1.7 Patients must be able to provide written informed consent 
3.1.8 Negative serum pregnancy test within 28 days prior to commencement of dosing in premenopausal 
women. Negative urine pregnancy test within 24 hours of starting treatment.  Women of non-
childbearing potential may be included without serum pregnancy test if they are either surgically 
sterile or have been postmenopausal for ≥ 1 year. Women and men must use an effective method 
of contraception from 14 days prior to start of treatment, throughout the treatment period, and for at 

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least 6 months after the last dose of study treatment as directed by their physician.  Effective 
methods of contraception are defined as those which result in a low failure rate (i.e., less than 1% 
per year) when used consistently and correctly (for example implants, injectables, or intra-uterine 
devices).  At the discretion of the investigator, acceptable methods of contraception may include 
total abstinence in cases where the lifestyle of the patient ensures compliance.  (Periodic 
abstinence [e.g., calendar, ovulation, symptothermal, postovulation methods] and withdrawal are 
not acceptable methods of contraception.) Hormonal-based methods (e.g., oral contraceptives) are 
permitted. 
3.1.9 Patients must be able to swallow and retain oral medication and must not have any clinically 
significant gastrointestinal abnormalities that may alter absorption such as malabsorption syndrome 
or major resection of the stomach or bowels. 
3.1.10 Patients must have adequate baseline organ function as determined by Table 1. 
Table 1. Definitions for adequate baseline organ function 
System 
Laboratory Values 
 
Hematologic 
Absolute neutrophil count (ANC) 
 1.0 × 109/L 
Hemoglobin 
 9 g/dL  
Platelet count 
 100 x 109/L 
PT/INRa and PTT 
 1.3 x ULN 
 
Hepatic 
Total bilirubinb 
 1.5 x ULN 
AST and ALT 
 2.5 x ULN  
 
Renal 
Serum creatininec 
 
 1.5 mg/dL 
 
Abbreviations: ALT = alanine transaminase; ANC = absolute neutrophil count; AST = aspartate 
aminotransferase; INR = international normalized ratio; LLN = lower limit of normal; PT = 
prothrombin time; PTT = partial thromboplastin time; ULN = upper limit of normal. 
a  Subjects receiving anticoagulation treatment may be allowed to participate with INR 
established within the therapeutic range prior to enrollment. 
b  Subjects with known Gilbert’s syndrome must have a total bilirubin < 3.0 x ULN). 
c  If serum creatinine is > 1.5 mg/dL, calculate creatinine clearance using standard Cockcroft-
Gault formula.  Creatinine clearance must be  50 mL/min to be eligible. 
 
3.2 Exclusion Criteria: 
3.2.1 Patients with known serious concurrent infection or medical illness, including psychiatric 
disorders, which would jeopardize the ability of the patient to receive the treatment outlined in this 
protocol with reasonable safety. 
3.2.2 Patients who are pregnant or breast-feeding. 
3.2.3 Patients who received prior anti LAG3 antibody are excluded  
3.2.4 Patients with brain metastases treated with whole brain radiation that have been stable for 2 
months are eligible; patients with brain metastases treated with gamma knife or surgery are 
allowed to participate after 2 weeks have elapsed since their procedure. Subjects are excluded if 

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they have leptomeningeal disease or metastases causing spinal cord compression that are 
symptomatic or untreated or not stable for ³ 3 months (must be documented by imaging) or 
requiring corticosteroids> 20 mg prednisone equivalent daily.  Subjects on a stable dose of 
corticosteroids > 1 month or who are tapering off steroids and have reached an equivalent of 20 
mg prednisone can be enrolled with approval of the Study PI. 
3.2.5 Patients must have discontinued active immunotherapy (IL-2, interferon, CTLA-4, etc.), 
chemotherapy, or investigational anticancer therapy at least 4 weeks prior to entering the study 
and oral targeted therapy at least 2 weeks prior to entering the study. 
3.2.6 All prior anti-cancer treatment-related toxicities (except alopecia and laboratory values as listed in 
3.1.10) must be ≤ Grade 1 or irreversible (hypophysitis) according to the Common Terminology 
Criteria for Adverse Events version 5 at the time of starting treatment. Patients that are 
asymptomatic on low dose maintenance hormone replacement delivered at a stable dose for prior 
toxicities are eligible. 
3.2.7 Patients who are known to be experiencing an objective partial response or stable disease  to 
immunotherapy at the time of study enrollment. 
3.2.8 History of malignancy other than disease under study within 3 years of study enrollment with 
exceptions below: Subjects with a history of completely resected non-melanoma skin cancer, or 
subjects with indolent second malignancies are eligible. 
3.2.9 Diagnosis of severe autoimmune disease requiring immunosuppressive medications. Patients 
with adrenal insufficiency on replacement dose steroids are eligible. 
3.2.10 History of interstitial lung disease or chronic pneumonitis unrelated to prior immunotherapy. Prior 
interstitial pneumonitis related to immunotherapy that was completely treated with no need for 
ongoing clinical management is allowed. 
3.2.11 Due to risk of disease exacerbation patients with porphyria or psoriasis are ineligible unless the 
disease is well controlled and they are under the care of a specialist for the disorder who agrees 
to monitor the patient for exacerbations. 
3.2.12 Have a known immediate or delayed hypersensitivity reaction or idiosyncrasy to drugs chemically 
related to study drug, or excipients or to dimethyl sulfoxide. 
3.2.13 Patients receiving cytochrome P450 enzyme-inducing anticonvulsant drugs (i.e. phenytoin, 
carbamazepine, Phenobarbital, primidone or oxcarbazepine) within 4 weeks of the start of the 
study treatment 
3.2.14 Current use of a prohibited medication as described in section on Potential for Drug-Drug 
Interaction. 
3.2.15 History or evidence of increased cardiovascular risk including any of the following: 
• 
Patients with a baseline troponin > 2XULN  
• 
Left ventricular ejection fraction (LVEF) < institutional lower limit of normal. Baseline 
echocardiogram is not required. 
• 
A QT interval corrected for heart rate using the Bazett‘s formula > 500 msec; 
• 
Current clinically significant uncontrolled arrhythmias. Exception: Subjects with controlled 
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• 
History of acute coronary syndromes (including myocardial infarction and unstable angina), 
coronary angioplasty, or stenting within 6 months prior to enrollment 
• 
Current ≥ Class II congestive heart failure as defined by New York Heart Association 
• 
History of myocarditis  
• 
 
 
4.0 TREATMENT PLAN 
This is a Phase 1/2 study (Fig. 3), in which the Phase 1a portion will identify the MTD of combination 
HCQ + nivolumab therapy in a cohort of subjects with anti-PD-1 Ab naïve or refractory melanoma. This 
will be followed by an open-label expansion in Cohort 2a: anti-PD-1 Ab refractory patients and Cohort 2b: 
anti-PD-1 Ab naïve stage III unresectable or stage IV melanoma patients. Cohort 2c: anti-PD-1 refractory 
Stage III unresectable or Stage IV melanoma.  
Once the phase 1a HCQ + nivolumab portion is completed and phase 2 is launched, the phase 1b HCQ 
+ nivolumab + ipilimumab portion of the study will begin accrual to identify the MTD of HCQ + nivolumab 
+ ipilimumab. Phase 1a HCQ + Nivolumab. The phase 1a dose-escalation will proceed via a standard 
3+3 dose-escalation design evaluating combination therapy at 2 dose levels of HCQ (400 mg BID and 
600 mg BID). 
Dose Level 1:  HCQ 400 mg PO twice daily + nivolumab 480 mg flat-dose IV every 4 weeks 
Dose Level 2:  HCQ 600 mg PO twice daily + nivolumab 480 mg flat-dose IV every 4 weeks 
Safety will be assessed beginning on day 1 of therapy and will continue until the completion of study 
follow-up.  Assessment for dose-limiting toxicity (DLT) in the Phase I portion will continue through the 
completion of 1 cycle of combination therapy (28 days).  A minimum of 3 subjects will be enrolled and 
treated in the first dose-level cohort, and all 3 subjects will be observed for a minimum of 28 days before 
subsequent cohort enrollment begins.  We anticipate proceeding rapidly through the dose-escalation 
portion of the study, given our prior experience with combination HCQ and multiple anti-cancer therapies 
 
Fig. 3 Modified LIMIT Melanoma Trial Schema. Dotted lines indicate Stage II of Simon 2-stage design Stage 
II cohort that would be activated if there is sufficient activity in Stage I.  

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in similar settings.  Following determination of the MTD, enrollment will proceed to the Phase II portion of 
the study.  The phase II cohort expansions will further explore the safety, preliminary efficacy, and 
important pharmacodynamic correlative information at the MTD combination regimen. 
Phase 2 Expansion Cohort Dosing:  MTD HCQ (400 or 600 mg PO twice daily) + nivolumab 480 mg 
flat-dose IV every 4 weeks 
Phase 2 Cohort a: Previously treated with anti-PD-1 Ab. In 2023 this cohort will be closed early due to 
developments in the Stage IV melanoma management that make this combination unlikely to be 
developed further.  
Phase 2 Cohort b: Anti-PD-1 Ab naïve melanoma. In 2023 this cohort will be closed early due to 
developments in the Stage IV melanoma management that make this combination unlikely to be 
developed further. 
Phase 2c cohort: Nivolumab-relatlimab and HCQ. HCQ 600 mg PO twice a daily + nivolumab 480 
mg + relatlimab 160 mg IV every 4 weeks  
Previously treated with Anti-PD-1 Ab.   
All trial treatments will be administered on an outpatient basis. Nivolumab, ipilimumab, and nivolumab-
relatlimab will be administered as 30-minute IV infusions. However given the variability of the infusion 
pumps, a window of -5 minutes to +10 minutes is allowed.  
CT scans will be obtained every 3 cycles (84 days or 12 weeks +/- 1 week) for assessment of disease 
response, as per RECIST 1.1 criteria.  Toxicity will be assessed using the NCI CTCAE criteria, version 
5.0.  Serial blood samples will be obtained for pharmacodynamic correlative assessments.  Radiology-
guided pre-treatment and on-treatment (cycle 1 day 12 +/- 2 days) metastatic core tissue sampling will be 
obtained for correlative pharmacodynamic and immunophenotyping studies. 
The treatment period for combination therapy will continue every 28 days for up to 24 months.  Safety 
follow-up will occur for approximately 100 days following the last administration of study treatment. 
Once the Phase 1a nivolumab + HCQ study is completed and the phase 2 cohorts are launched, in 
parallel the phase 1b nivolumab + ipilimumab + HCQ study will begin accrual (Fig. 3). The Phase I (3-
drug combination) trial will identify the MTD of combination HCQ + nivolumab 1 mg/kg i.v. every 3 weeks 
x4 in combination with ipilimumab 3 mg/kg i.v. every 3 weeks X 4 cycles followed by maintenance 
nivolumab therapy in patients with anti-PD-1 Ab refractory melanoma. 
 
Phase 1b nivolumab + ipilimumab + HCQ. In the phase 1b nivolumab + ipilimumab + HCQ (3-drug 
combination) trial, dose-escalation will proceed via a standard 3+3 dose-escalation design evaluating 
combination therapy at 2 dose levels of HCQ (400 mg BID and 600 mg BID). 
Dose Level 1:  HCQ 400 mg PO BID + nivolumab 1 mg/kg IV given over 30 minutes , followed by 
ipilimumab 3 mg/kg IV given over 30 minutes. The nivolumab + ipilimumab infusions will be given every 3 
weeks X 4 administrations followed by maintenance nivolumab 480 mg IV every 4 weeks until 
progression or 24 months.  Maintenance nivolumab will be started 6 weeks after the last dose of 
ipilimumab and nivolumab. Patients who discontinue combination ipilimumab and nivolumab for Grade 3-
4 toxicity will not be treated with maintenance nivolumab or HCQ, unless the grade 3-4 toxicity is easily 
managed and completely resolved. These patients will be evaluable for response, PFS and toxicity on 
study. 
Dose Level 2:  HCQ 600 mg PO BID + the same nivolumab + ipilimumab combination, dosing, schedule, 
and specifications as dose level 1 
Dose Level 1.5 HCQ 400 mg AM/600 mg PM po BID + the same nivolumab + ipilimumab combination, 
dosing, schedule, and specifications as dose level 1 

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Safety will be assessed beginning on day 1 of therapy and will continue until the completion of study 
follow-up.  Assessment for dose-limiting toxicity (DLT) in the Phase I portion will continue through the 
completion of 1 cycles of combination therapy (21 days).  A minimum of 3 subjects will be enrolled and 
treated in the first dose-level cohort, and all 3 subjects will be observed for a minimum of 21 days before 
subsequent cohort enrollment begins.  We anticipate proceeding rapidly through the dose-escalation 
portion of the study, given our prior experience with combination HCQ and multiple anti-cancer therapies 
in similar settings. 
5.0 DETAILS OF STUDY TREATMENT 
5.1 Hydroxychloroquine 
Mechanism of Action: The mechanism of action is not fully understood. Previously it was thought that 
HCQ and other chloroquine derivatives are weak bases that deacidify lysosomes through purely chemical 
basis. Recently our group has identified the missing molecular target of HCQ as palmitoyl protein 
thioesterase 1 (PPT1). 
Storage and formulation: HCQ tablets are manufactured by a number of generic drug companies. Each 
tablet contains 200 mg hydroxychloroquine sulfate (equivalent to 155 mg base). It is dispensed in a tight, 
light-resistant container as defined in the USP/NF.HCQ should be stored at room temperature up to 30° 
C (86° F). 
Pharmacokinetics: The PK of HCQ is characterized by a large volume of distribution, binding to red blood 
cells, and long time to peak concentration and steady state. Population PK studies in cancer patients 
have demonstrated dose proportional change in exposure. 
Administration: Hydroxychloroquine is an oral medication, requiring the patients on study to keep a study 
diary. Diary must be submitted at each clinic visit. Hydroxychloroquine should be obtained commercially.  
The starting dose for HCQ will be 400 mg po twice daily continuously. Tablets of HCQ are available in 
200 mg strength. HCQ will be administered in divided doses (every 12 hours) Take hydroxychloroquine 
sulfate tablets with a meal or a glass of milk. The two daily doses of HCQ should be taken 12 hours 
apart, for example, 9 am and 9 pm, and documented clearly on the patient calendar.  The HCQ 
schedule may be adjusted if necessary to minimize gastrointestinal side effects. 
For complete information please refer to the package inserts at http://dailymed.nlm.nih.gov/dailymed/ 
 
 
5.2 Nivolumab 
Mechanism of Action: Binding of the PD-1 ligands, PD-L1 and PD-L2, to the PD-1 receptor found on T-
cells, inhibits T-cell proliferation and cytokine production. Upregulation of PD-1 ligands occur in some 
tumors and signaling through this pathway can contribute to inhibition of active T-cell immune 
surveillance of tumors. Nivolumab is a human immunoglobulin G4 monoclonal antibody that binds to the 
PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, releasing the PD-1 pathway-mediated 
inhibition of the immune response, including the anti-tumor immune response. 
Storage and formulation: Nivolumab currently comes packaged in a concentrated form in 100 mg (10 
mg/mL) single use vial. This concentrated solution should be diluted with 0.9% Sodium Chloride or 5% 
Dextrose Injection to prepare a diluted solution with a final concentration ranging from 1 mg/mL to 10 
mg/mL. Nivolumab must be stored at 2°-8°C. Vials should be protected from light and should not be 
frozen. 
Pharmacokinetics: The pharmacokinetics of nivolumab was studied in 909 patients over a dose range of 
0.1 to 20 mg/kg administered as a single dose or as multiple doses every 2-4 weeks. The exposure to 
nivolumab increased dose proportionally over the dose range of 0.1 to 10 mg/kg administered every 2 
weeks. The mean CL is 9.5 mL/h and mean elimination half-life is 25.7 days. Steady-concentrations of 

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nivolumab were reached by 12 weeks when administered at 3 mg/kg every 2 weeks and systemic 
accumulation was approximately 3-fold. 
Administration: Nivolumab will be mixed, stored, and administered intravenously as per investigators 
brochure. 
5.3 Ipilimumab 
Mechanism of Action: Ipilimumab is a monoclonal antibody, more specifically a fully humanized IgG1 
antibody produced in mammalian cell culture, to the cytotoxic T lymphocyte antigen-4 (CTLA-4) which 
activates antitumor immunity by inhibiting this major checkpoint 
Storage and formulation: Ipilimumab is supplied and stored as a 50-mg single-use 10 mL vial.  
Preparation of solution: Allow the vials to stand at room temperature for approximately 5 minutes prior to 
preparation of infusion. Withdraw the required volume of ipilimumab and transfer into an intravenous bag. 
Dilute with 0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, USP to prepare a diluted 
solution with a final concentration ranging from 1 mg/mL to 2 mg/mL. Mix diluted solution by gentle 
inversion. Store the diluted solution for no more than 24 hours under refrigeration (2°C to 8°C, 36°F to 
46°F) or at room temperature (20°C to 25°C, 68°F to 77°F). Discard partially used vials or empty vials. 
Administration instructions. Do not mix YERVOY with, or administer as an infusion with, other medicinal 
products. Flush the intravenous line with 0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, 
USP after each dose. Administer diluted solution over 30 minutes through an intravenous line containing 
a sterile, non-pyrogenic, low-protein–binding in-line filter. 
Pharmacokinetics: The pharmacokinetics (PK) of ipilimumab were studied in 785 patients with 
unresectable or metastatic melanoma who received doses of 0.3, 3, or 10 mg/kg once every 3 weeks for 
4 doses. The PK of ipilimumab is linear in the dose range of 0.3 to 10 mg/kg. Following administration of 
YERVOY every 3 weeks, the systemic accumulation was 1.5-fold or less. Steady-state concentrations of 
ipilimumab were reached by the third dose; the mean Cmin at steady state was 19.4 mcg/mL at 3 mg/kg 
and 58.1 mcg/mL at 10 mg/kg every 3 weeks. The mean value (percent coefficient of variation) based on 
population PK analysis for the terminal half-life (t1/2) was 15.4 days (34%) and for clearance (CL) was 
16.8 mL/h (38%).YERVOY with nivolumab: When ipilimumab 1 mg/kg was administered in combination 
with nivolumab 3 mg/kg, the CL of ipilimumab and nivolumab were unchanged compared to when 
YERVOY was administered alone. When administered in combination, the CL of ipilimumab was 
unchanged in presence of anti-ipilimumab antibodies and the CL of nivolumab increased by 20% in the 
presence of anti-nivolumab antibodies. 
Administration: Ipilimumab will be mixed, stored and administered intravenously as per package insert. 
5.4 Nivolumab-relatlimab:  
Mechanism of Action: Relatlimab is a human IgG4 monoclonal antibody that binds to the LAG-3 receptor, 
blocks interaction with its ligands, including MHC II, and reduces LAG-3 pathway-mediated inhibition 
of the immune response. Antagonism of this pathway promotes T cell proliferation and cytokine 
secretion.  
 
Binding of the PD-1 ligands, PD-L1 and PD-L2, to the PD-1 receptor found on T cells, inhibits 
T-cell proliferation and cytokine production. Upregulation of PD-1 ligands occurs in some tumors, 
and signaling through this pathway can contribute to inhibition of active T-cell immune 
surveillance of tumors. Nivolumab is a human IgG4 monoclonal antibody that binds to the PD-1 
receptor, blocks interaction with its ligands PD-L1 and PD-L2, and reduces PD-1 pathway- 
mediated inhibition of the immune response, including the anti-tumor immune response. In 
syngeneic mouse tumor models, blocking PD-1 activity resulted in decreased tumor growth. 
The combination of nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) results in increased T-cell 
activation compared to the activity of either antibody alone.  

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In murine syngeneic tumor models,LAG-3 blockade potentiates the anti-tumor activity of PD-1 blockage, 
inhibiting tumor growth and promoting tumor regression. 
 
Storage and formulation: OPDUALAG is a fixed-dose combination of nivolumab and relatlimab. 
Visually inspect the solution in the drug product vial for particulate matter and discoloration prior 
to administration. OPDUALAG is a clear to opalescent, colorless to slightly yellow solution. 
Discard the vial if the solution is cloudy, discolored, or contains extraneous particulate matter other 
than a few translucent-to-white particles.  
 
Preparation: 
• During preparation of the infusion solution, use aseptic technique to assure sterility, as the 
product does not contain a preservative. 
• OPDUALAG can be administered diluted or undiluted and administered at a final 
concentration as specified in the package insert  
• Withdraw the required volume of OPDUALAG and transfer into an intravenous container. 
OPDUALAG is compatible with di(2-ethylhexyl)phthalate (DEHP)-plasticized polyvinyl 
chloride (PVC), ethyl vinyl acetate (EVA), and polyolefin (PO) intravenous bags. 
• If diluting OPDUALAG prior to administration: 
− Dilute OPDUALAG solution with 0.9% Sodium Chloride Injection, USP or 5% 
Dextrose Injection, USP to prepare an infusion meeting the final concentration and 
maximum infusion volume parameters as specified in Table 2 below. 
− Then mix the diluted solution by gentle inversion. Do not shake. 
• Discard partially used vials or empty vials following infusion preparation. 
* The concentration range in each group includes 12 mg/mL nivolumab and 4 mg/mL relatlimab as the 
upper limit, 
which represents a scenario in which the drug product is infused without dilution. 
 
Storage of Prepared Solution: 
Store the prepared solution either: 
• at room temperature and room light for no more than 8 hours from the time of preparation 
to the end of the infusion. Discard the prepared solution if not used within 8 hours from the 
time of preparation; 
-or- 
• under refrigeration at 2°C to 8°C (36°F to 46°F) with protection from light for no more 
than 24 hours from the time of preparation, which includes the time allowed for 
equilibration of the infusion bag to room temperature and the duration of the infusion. 
Discard the prepared solution if not used within 24 hours from the time of preparation. 
Do not freeze. 
 
Administration: 
• Administer the infusion over 30 minutes through an intravenous line containing a sterile, 
non-pyrogenic, low protein binding in-line polyethersulfone (PES), nylon, or 
polyvinylidene fluoride (PVDF) filter (pore size of 0.2 micrometer to 1.2 micrometer). 
• Flush the intravenous line at the end of the infusion. 
• Do not coadminister other drugs through the same intravenous line. 
 
Pharmacokinetics: The pharmacokinetics (PK) of relatlimab following the administration of OPDUALAG 
were characterized in patients with cancer who received relatlimab 20 to 800 mg every 2 weeks (0.25 
to 10 times the approved recommended dosage) or 160 to 1440 mg every 4 weeks (1 to 9 times 
the approved recommended dosage) either as a monotherapy or in combination with nivolumab 
dosages of 80 or 240 mg every 2 weeks or 480 mg every 4 weeks. 

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Steady-state concentrations of relatlimab were reached by 16 weeks with an every 4-week regimen 
and the systemic accumulation was 1.9-fold. The average concentration (C avg) of relatlimab after 
the first dose increased dose proportionally at doses ≥160 mg every 4 weeks. 
Following the recommended dosage, the geometric mean [coefficient of variation (CV%)] 
maximum and average concentrations (Cmax and Cavg) of relatlimab at steady state were 62.2 
(30%), and 28.8 (45%) μg/mL, respectively; and the mean Cmax and Cavg of nivolumab at steady 
state were 187 (33%) and 94.4 (43%) μg/mL, respectively. 
In RELATIVITY-047, the nivolumab geometric mean minimum concentration (Cmin ) at steady 
state in the OPDUALAG arm was comparable to the nivolumab arm. 
 
Distribution: 
The geometric mean (CV%) volume of distribution at steady state of relatlimab is 6.6 L (20%) and 
6.6 L (19%) of nivolumab. 
 
Elimination: 
The geometric mean (CV%) clearance of relatlimab is 5.5 mL/h (41%) at steady state, 10% lower 
than after the first dose [6 mL/h (39%)]. Following OPDUALAG (nivolumab 480 mg and 
relatlimab 160 mg administered every 4 weeks) administration, the geometric mean (CV%) 
effective half-life (t1/2 ) of relatlimab is 26.2 days (37%). The geometric mean (CV%) clearance of 
nivolumab is 7.6 mL/h (40%) at steady state, 21% lower 
than after the first dose [9.6 mL/h (40%)] and the terminal t1/2 is 26.5 days (36%). 
 
Specific Populations: 
The following factors had no clinically important effect on the clearance of nivolumab and 
relatlimab: age (17 to 92 years), sex, race (White, Asian, and Black/African American), mild or 
moderate renal impairment (eGFR 30 to 89 mL/min/1.73 m 2 ), mild hepatic impairment (total 
bilirubin [TB] less than or equal to upper limit of normal [ULN] and AST greater than ULN or TB 
greater than 1 to 1.5 times ULN and any AST) or moderate hepatic impairment (TB greater than 
1.5 to 3 times ULN and any AST). The effects of severe renal impairment, or severe hepatic 
impairment on the pharmacokinetics of nivolumab and relatlimab are unknown. 
 
5.5 Concomitant Medication, Drug-Drug interactions and Procedures 
Subjects must be instructed not to take any medications, including over the counter products, without first 
consulting with the investigator. 
Because HCQ has known effects on P450 enzymes, patients requiring anti-convulsants may be treated 
with any of the non-enzyme inducing anti-convulsants which include: felbamate, valproic acid, 
gabapentin, lamotrigine, tiagibine, topiramate, or levetiracetam. Due to the fact that both zonisamide and 
HCQ accumulate in red blood cells, zonisamide should be avoided if possible. For nausea, aprepitant 
should be avoided. Radiation therapy to the surgical bed with gamma knife radiotherapy while on 
treatment is allowed for surgically resected brain metastases. Gamma knife for new CNS lesions may be 
performed while on study, but further of treatment of these patients will be considered treatment beyond 
progression.  All other concomitant medications are permitted. 
The following medications are not allowed during the study. The sponsor must be notified if the subject 
receives any of these during the study: 
1. Any investigational anticancer therapy 
2. Any concurrent chemotherapy, radiotherapy (except radiotherapy as designated in this study or 
radiotherapy indicated for CNS metastasis), immunotherapy, biologic or hormonal therapy for cancer 
treatment, except as noted in the exclusion criteria. Concurrent use of hormones for noncancer-related 
conditions (e.g. insulin for diabetes, hormone replacement therapy) is acceptable. 

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3. Immunosuppressive medications, including, corticosteroids at doses exceeding 10mg/day of 
prednisone or equivalent, methotrexate, azathioprine, and TNF-alpha blockers. Use of 
immunosuppressive medication for the management of study treatment-related AEs or in subjects with 
contrast allergies is acceptable. In addition, use of topical, inhaled and intranasal corticosteroids is 
permitted 
4. Live attenuated vaccines during the study through 180 days after the last dose of both drugs 
5. Herbal and natural remedies should be avoided 
5.6 Duration of Protocol Treatment and Follow-up 
Treatment will continue until disease progression or unacceptable toxicity for a maximum of 24 months 
for patients with partial or complete response. For patients experiencing stable disease treatment may 
continue at the discretion of the treating physician in consultation with the Study Chair and Medical 
Monitor. Treatment may also be continued for isolated progression that can be treated with local therapy.  
For patients that experience a complete response treatment (including HCQ) can be discontinued after 9 
months of combined therapy. Subjects will be evaluated for at least 100 days after discontinuation of 
study treatment and will be followed for a minimum of one year from the start of treatment, unless 
disease progression is reported prior to one year.  One-year survival information will be extracted from 
the medical record or by phone call for subjects who have discontinued protocol treatment and completed 
the 100 day follow-up evaluations prior to one year from the start of treatment. 
 
 
6.0 DOSE LIMITING TOXICITY AND DOSE ESCALATION RULES 
6.1 Definition of Dose Limiting Toxicity (DLT) 
For phase 1a nivolumab and HCQ, if occurring in the first 4 weeks of combined therapy the following will 
be considered DLTs: 
1. grade 4 neutropenia with fever 
2. grade 4 thrombocytopenia 
3. Any non-hematologic toxicity of grade 3 or higher that is at least possibly treatment-related and 
that is refractory to supportive measures. These include but are not limited to fatigue, nausea, 
diarrhea, visual disturbance. This excludes nausea and vomiting which have not been treated with 
optimal anti-emetic therapy.  Grade 3 rash can also occur with nivolumab, and therefore will not be 
considered a DLT, unless the quality of the rash is more severe than what would be expected with 
nivolumab alone. 
For Phase I nivolumab, ipilimumab and HCQ, rash, fatigue and gastrointestinal adverse events are 
common to nivolumab, Ipilimumab and high dose HCQ. Since grade 3 rash, fatigue and diarrhea can 
occur with nivolumab, ipilimumab or the combination in the first 3 weeks a grade 3 will be considered a 
DLT in this trial only if maximal supportive measures are refractory and rash does not improve with 
steroids.  Grade 3 non-hematological toxicities not common to all of the three drugs that are refractory to 
maximal supportive care will be considered DLTs. 
Any DLT that causes a patient to miss > 28 consecutive days of HCQ will result in the patient being taken 
off HCQ treatment. Patients will continue on nivolumab or nivolumab and ipilimumab if that is appropriate 
at that point and remain evaluable for toxicity and response as long as they received at least 4 weeks of 
combined nivolumab + HCQ prior to discontinuation of HCQ. 

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6.2 Dose Escalation Rules 
The target dose limiting toxicity (DLT) rate is ≤ 33%.  The MTD is defined as a) the dose producing DLT 
in 2 out of 6 patients, or b) the dose level below the dose which produced DLT in ≥ 2 out of 3 patients, or 
in ≥ 3 out of 6 patients.  No intra-patient dose escalation will be permitted. Patients will be evaluable for a 
Phase I cohort if they completed 75% of their expected dose of HCQ for the first cycle. 
The first three patients will be enrolled on dose level 1 as described in Table 3. If a DLT is observed in 1 
patient out of 3 the cohort will be expanded to 6. If a DLT occurs in 2 out of 3 or more patients per cohort, 
then the cohort one dose below will be the declared MTD provided that at least 6 patients have been 
treated at that level with no more than 2 having DLTs. The rules for dose escalation and cohort size are 
outlined in Table 2. No intra-patient dose escalation is planned. Patients will be evaluable for DLT if they 
have completed 75% of expected HCQ dose for combined HCQ and nivolumab or HCQ + nivolumab + 
ipilimumab during the first cycle of therapy. 
Table 2:  Criteria for Dose Escalation and Cohort Size 
 
# with 
DLT 
At dose level 1 
At dose level 2 
At dose level 1.5 
0/3 
Escalate to dose level 
2 
Declare dose level 2 the  
RP2D  
Declare dose level 1.5 the 
RP2D 
1/3 
Add 3 patients 
Add 3 patients 
Add 3 patients 
>2/3 
Close trial 
De-escalate to dose level 1.5 Declare dose level 1 MTD 
  
If 6 are enrolled: 
  
  
1/6 
Escalate to dose level 
2 
Declare dose level 2 MTD 
Declare dose level 1.5 MTD 
2/6 
Close trial  
De-escalate to dose level 1.5 Declare Dose level 1 MTD 
RP2D: Recommended phase II dose 
 
6.3 Dose Levels 
Table 3: Hydroxychloroquine plus Nivolumab Dose Escalation Schema 
 
Dose level 
Nivolumab  
HCQ 
1 
480 mg q4 weeks 
400 mg bid  
1.5 
480 mg q4 weeks 
400 mg am / 600 mg pm 
2 
480 mg q4 weeks 
600 mg bid 
 
Table 4: Hydroxychloroquine plus Nivolumab and Ipilimumab Dose Escalation Schema 
 
Dose level 
Nivolumab  
Ipilimumab 
HCQ 
1 
1 mg/kg IV q 3weeks x4 
3 mg/kg IV q 3 weeks x4 400 mg bid  
1.5 
1 mg/kg IV q 3 weeks x4 3 mg/kg IV q 3 weeks x4 400 mg am / 600 mg pm 
2 
1 mg/kg IV q 3 weeks x4 3 mg/kg IV q 3 weeks x4 600 mg bid 
 
 

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6.4 Single arm Phase 2 trial with 2 cohorts 
Once the MTD or recommended phase II dose for HCQ when given in combination with nivolumab is 
determined in the phase I portion of this trial, a non- randomized 2 cohort single arm open label phase II 
trial will be conducted. The trial is designed using a Simon 2 stage phase II trial approach (see Section 
12.2), however, accrual will not be halted until enough patients in the first stage are evaluable for the 
primary endpoint (response) to trigger the stopping rule. 
7.0 TOXICITY CRITERIA, MONITORING, DOSE DELAYS AND MODIFICATIONS 
7.1 Toxicity Criteria 
This study will utilize the CTCAE version 5.0 for toxicity and Adverse Event Reporting.  A copy of the 
CTCAE version 5.0 can be downloaded from the CTEP home page 
(https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm). All appropriate treatment 
areas should have access to a copy of the CTCAE version 5.0. 
7.2 Dose Delays 
Major Events are grade 3 and 4 hematologic and non-hematologic toxicities that are not treatment -
related. Treatment should be delayed for major events if HCQ may further complicate the non-treatment 
related event.  If a major event requires a delay of treatment, treatment must be delayed until toxicity is 
resolved (≤ Grade 2 or ≤ Baseline). For treatment-related toxicities and major events, if toxicity 
(exceptions are endocrine and mild skin toxicities) is not resolved in ≤ 28 days, patient will be taken off 
treatment, unless there is an exception granted by the medical monitor. 
7.3 Ocular toxicity 
The only toxicity that requires discontinuation of HCQ is retinopathy. Published literature indicates HCQ 
retinopathy is idiosyncratic and is uncommon in patients receiving HCQ for less than a few years. Our 
ongoing study of dabrafenib, trametinib, and HCQ (BAMM; NCTNCT02257424) found no clinically 
meaningful ocular toxicity in 10 patients studied extensively with serial ocular exams 38. Therefore, we 
have not included mandatory ocular exams in this protocol. However, if there is a visual field deficit, 
retinal vein occlusion, serous retinopathy, bullseye retinopathy, or retinal detachment, HCQ should be 
permanently discontinued. 
7.4 Nivolumab Side Effects Overview 
Common Side Effects: Diarrhea, inflammation of the colon (colitis), increase in liver enzymes, fatigue, 
swelling of the extremities, flu-like feeling, pain, skin itchiness, skin rash or xerosis (increased 
dryness), vitiligo (loss of pigment or color in the skin), nausea, abdominal pain, decreased appetite, 
fever, vomiting, constipation, flatulence, dry mouth, weight loss, cough, shortness of breath, 
headache, peripheral neuropathy (numbness or tingling of the fingers or toes), dizziness, change in 
sensation of taste, joint pain or stiffness, dehydration, increased blood sugar, low sodium levels, 
infections, inflammation of the optic disc, low blood pressure, and difficulty sleeping. 
Less Likely Side Effects: inflammation of the pancreas, decreased movement of the intestines, 
inflammation of the thyroid, lung inflammation (pneumonitis), shortness of breath, joint swelling, 
muscle soreness, weakness, stiffness, or spasm, adrenal gland abnormalities, inflammation of the 
heart or lining of the heart, acute kidney injury or failure, and increase in inflammatory blood proteins. 
Rare Side Effects: 
• 
Myasthenia gravis, a nerve disease that may cause weakness of the eye, face, breathing and 
swallowing muscles. One death in a patient who received the combination was considered 
due to myasthenia gravis and severe infection (sepsis) 
• 
A syndrome associated with fever, WBC activation and abnormal function (including destruction 
of other blood cells by WBCs), low blood cell counts, rash, and enlargement of the spleen. 

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7.5 Treatment of Nivolumab, Nivolumab +Ipilimumab, or nivolumab-relatlimb Related Infusion 
Reactions 
Since nivolumab contains only human immunoglobulin protein sequences, it is unlikely to be 
immunogenic and induce infusion or hypersensitivity reactions. However, if such a reaction were to 
occur, it might manifest with fever, chills, rigors, headache, rash, pruritus, arthralgias, hypo- or 
hypertension, bronchospasm, or other symptoms. 
All Grade 3 or 4 infusion reactions should be reported as serious adverse events (SAEs) if criteria are 
met. Infusion reactions should be graded according to National Cancer Institute (NCI) CTCAE 5.0 
guidelines. 
Treatment recommendations are provided below and may be modified based on local treatment 
standards and guidelines as appropriate: 
 
For Grade 1 symptoms: (Mild reaction; infusion interruption not indicated; intervention not indicated). 
Remain at bedside and monitor patient until recovery from symptoms. The following prophylactic 
premedications are recommended for future infusions: diphenhydramine 50 mg (or equivalent) and/or 
acetaminophen 325 to 1000 mg at least 30 minutes before additional nivolumab administrations. 
For Grade 2 symptoms: (Moderate reaction requires therapy or infusion interruption but responds promptly 
to symptomatic treatment [e.g., antihistamines, non-steroidal anti-inflammatory drugs, narcotics, 
corticosteroids, bronchodilators, intravenous (IV) fluids]; prophylactic medications indicated for 24 hours). 
Stop the nivolumab infusion, begin an IV infusion of normal saline, and treat the patient with 
diphenhydramine 50 mg IV (or equivalent) and/or acetaminophen 325 to 1000 mg; remain at bedside and 
monitor patient until resolution of symptoms. Corticosteroid or bronchodilator therapy may also be 
administered as appropriate. If the infusion is interrupted, then restart the infusion at 50% of the original 
infusion rate when symptoms resolve; if no further complications ensue after 30 minutes, the rate may be 
increased to 100% of the original infusion rate. Monitor patient closely. If symptoms recur then no further 
nivolumab will be administered at that visit. Administer diphenhydramine 50 mg IV, and remain at bedside 
and monitor the patient until resolution of symptoms. The amount of study drug infused must be recorded 
on the eCRF. The following prophylactic premedications are recommended for future infusions: 
diphenhydramine 50 mg (or equivalent) and/or acetaminophen 325 to 1000 mg should be administered at 
least 30 minutes before additional nivolumab administrations. If necessary, corticosteroids (recommended 
dose: up to 25 mg of IV hydrocortisone or equivalent) may be used. 
For Grade 3 or Grade 4 symptoms: (Severe reaction, Grade 3: prolonged [i.e., not rapidly responsive to 
symptomatic medication and/or brief interruption of infusion]; recurrence of symptoms following initial 
improvement; hospitalization indicated for other clinical sequelae [e.g., renal impairment, pulmonary 
infiltrates]). Grade 4: (life threatening; pressor or ventilatory support indicated). Immediately discontinue 
infusion of nivolumab or ipilimumab. Begin an IV infusion of normal saline, and treat the patient as follows. 
Recommend bronchodilators, epinephrine 0.2 to 1 mg of a 1:1,000 solution for subcutaneous 
administration or 0.1 to 0.25 mg of a 1:10,000 solution injected slowly for IV administration, and/or 
diphenhydramine 50 mg IV with methylprednisolone 100 mg IV (or equivalent), as needed. Patient should 
be monitored until the Investigator is comfortable that the symptoms will not recur. Nivolumab will be 
permanently discontinued. Investigators should follow their institutional guidelines for the treatment of 
anaphylaxis. Remain at bedside and monitor patient until recovery from symptoms. In the case of late-
occurring hypersensitivity symptoms (e.g., appearance of a localized or generalized pruritus within 1 week 
after treatment), symptomatic treatment may be given (e.g., oral antihistamine, or corticosteroids). 
 

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7.6 Ipilimumab Side Effects Overview 
Common Side Effects: Diarrhea, inflammation of the colon (colitis), increase in liver enzymes, fatigue, skin 
itchiness, skin rash, nausea, abdominal pain, decreased appetite, fever, vomiting, headache, constipation, 
adrenal gland abnormalities, and thyroid gland abnormalities. 
Less Likely Side Effects: Chills, weakness, muscle pain, and redness of skin. 
Rare Side Effects: Decrease or total loss of hormones of pituitary gland, allergic reactions, inflammation of 
the liver, inflammation of the pituitary gland, decreased red blood cells, loss of color (pigment) from areas 
of skin, decreased or blurry vision, or inflammation of the eye, numbness or tingling in your fingers or toes, 
inflammation or loss of the lining of the brain or spinal cord, inflammation of the kidneys, and joint pain. 
 
7.7 Ipilimumab and Nivolumab 
Common Side Effects: Diarrhea, inflammation of the colon (colitis), increase in liver enzymes, fatigue, 
swelling of the extremities, flu-like feeling, pain, skin itchiness, skin rash or xerosis (increased dryness), 
vitiligo (loss of pigment or color in the skin), nausea, abdominal pain, decreased appetite, fever, vomiting, 
constipation, flatulence, dry mouth, weight loss, cough, shortness of breath, headache, peripheral 
neuropathy (numbness or tingling of the fingers or toes), dizziness, change in sensation of taste, joint pain 
or stiffness, dehydration, increased blood sugar, low sodium levels, infections, inflammation of the optic 
disc, low blood pressure, and difficulty sleeping. 
Less Likely Side Effects: inflammation of the pancreas, decreased movement of the intestines, 
inflammation of the thyroid, lung inflammation (pneumonitis), shortness of breath, joint swelling, muscle 
soreness, weakness, stiffness, or spasm, adrenal gland abnormalities, inflammation of the heart or lining of 
the heart, acute kidney injury or failure, and increase in inflammatory blood proteins. 
Rare Side Effects: Myasthenia gravis, a nerve disease that may cause weakness of the eye, face, breathing 
and swallowing muscles. One death in a patient who received the combination was considered due to 
myasthenia gravis and severe infection (sepsis) 
A syndrome associated with fever, WBC activation and abnormal function (including destruction of other 
blood cells by WBCs), low blood cell counts, rash, and enlargement of the spleen. 
 
7.8 Nivolumab and relatlimab  
 
Very common (may affect more than 1 in 10 people) 
 
• 
Infection of the urinary tract (the parts of the body that collect and pass out urine) 
 
• 
Decreased number of red blood cells (which carry oxygen) and white blood cells (lymphocytes, 
neutrophils, leucocytes; which are important in fighting infection)  
• 
Underactive thyroid gland (which can cause tiredness or weight gain) 
 
• 
Decreased appetite 
 
• 
Headache 
 
• 
Difficulty breathing, cough 
• 
Diarrhoea (watery, loose or soft stools), vomiting; nausea; stomach pain; constipation 
 
• 
Skin rash (sometimes with blisters), skin colour change in patches (vitiligo), itching 
 

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• 
Pain in the muscles, bones and joints 
 
• 
Feeling tired or weak, fever. 
 
• 
Changes in the results of tests: 
abnormal liver function (increased amounts of the liver enzymes alkaline phosphatase, aspartate 
aminotransferase, alanine aminotransferase in your blood) 
abnormal kidney function (increased amounts of creatinine in your blood) 
decrease of sodium and magnesium, and decrease or increase of calcium and potassium. 
 
Common (may affect up to 1 in 10 people) 
 
• 
Infections of the upper respiratory tract (nose and upper airways) 
 
• 
Decreased number of platelets (cells which help the blood to clot), increase in some white blood 
cells 
 
• 
Decreased secretion of hormones produced by adrenal glands (glands situated above the 
kidneys), inflammation of the pituitary gland situated at the base of the brain, overactive thyroid 
gland, inflammation of the thyroid gland 
 
• 
Diabetes, low sugar levels in the blood; weight loss, high levels of the waste product uric acid in 
the blood, decreased levels of the protein albumin in the blood, dehydration 
state of confusion 
 
• 
Inflammation of the nerves (causing numbness, weakness, tingling or burning pain of the arms 
and legs), dizziness, changes in the sense of taste 
 
• 
Inflammation of the eye (which causes pain and redness, vision problems or blurry vision), 
vision problems, dry eyes, excessive tear production 
• 
Inflammation of the heart muscle 
 
• 
Inflammation of a vein, which can cause redness, tenderness and swelling 
Inflammation of the lungs (pneumonitis), characterised by coughing and difficulty breathing; 
 
• 
Nasal congestion (blocked nose) 
 
• 
Inflammation of the intestines (colitis), inflammation of the pancreas, inflammation of the 
stomach (gastritis), difficulty swallowing, mouth ulcers and cold sores; dry mouth 
inflammation of the liver (hepatitis) 
 
• 
Unusual hair loss or thinning (alopecia), isolated area of skin growth that becomes red and itchy 
(lichenoid keratosis), sensitivity to light, dry skin 
 
• 
Painful joints (arthritis), muscle spasms, muscle weakness 
 
• 
Kidney failure (changes in amount or colour of urine, blood in urine, swelling ankles, loss of 
appetite), high levels of proteins in the urine 
 

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• 
Edema (swelling), flu-like symptoms, chills 
 
• 
Reactions related to the administration of the medicine. 
 
• 
Changes in the results of tests carried out by your doctor may show: 
- abnormal liver function (higher blood levels of the waste product bilirubin, higher blood levels 
of the liver enzyme gamma-glutamyl transferase) 
- increase in sodium and magnesium 
- increased level of troponin (a protein released into the blood when the heart is damaged) 
- increased level of the enzyme that breaks down glucose (sugar) (lactate dehydrogenase), the 
enzyme that breaks down fats (lipase), the enzyme that breaks down starch (amylase) 
 
Uncommon (may affect up to 1 in 100 people) 
 
• 
Inflammation and infection in the hair follicles 
disorder in which red blood cells are destroyed faster than they can be made (haemolytic 
anaemia) 
 
• 
Underactive function of the pituitary gland situated at the base of the brain; underactive function 
of the glands producing sex hormones 
inflammation of the brain, which may include confusion, fever, memory problems or seizures 
(encephalitis), a temporary inflammation of the nerves that causes pain, weakness, and paralysis 
in the extremities (Guillain-Barré syndrome), inflammation of the optic nerve that may cause a 
complete or partial loss of vision 
 
• 
Inflammation of  the eyes, skin, in and the membranes of the ears, brain and 
spinal cord (Vogt-Koyanagi-Harada disease), red eye 
 
• 
Fluid around the heart 
 
• 
Asthma 
 
• 
Inflammation of the oesophagus (passage between throat and stomach) 
 
• 
Inflammation of the bile duct 
 
• 
Skin rashes and blistering on the legs, arms, and abdomen (pemphigoid), skin disease with 
thickened patches of red skin, often with silvery scales (psoriasis), hives (itchy, bumpy rash) 
inflammation of the muscles causing weakness, swelling, and pain, disease in which the 
immune system attacks the glands that make moisture for the body, such as tears and saliva 
(Sjogren’s syndrome), inflammation of muscles causing pain or stiffness, inflammation of the 
joints (painful joint disease), disease in which the immune system attacks its own tissues, 
causing widespread inflammation and tissue damage in the affected organs, such as joints, skin, 
brain, lungs, kidneys, and blood vessels (systemic lupus erythematosus) 
 
• 
Inflammation of the kidney 
 
• 
Absence of sperm in the semen. 
 

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• 
Changes in the results of tests carried out by your doctor may show: 
increase in level of c-reactive protein 
red blood cell sedimentation rate increased. 
 
 
 
7.9 Monitoring and Dose Delay Criteria 
Patients should be assessed for toxicity prior to each dose; dosing will occur only if the clinical assessment 
and laboratory test values are acceptable. If toxicity occurs as indicated below, patients in both study arms 
should have study drug(s) held. In patients who are in the combined treatment arm, both drugs should be 
held for any toxicity. Dose delays are designed to maximize treatment for those who derive clinical benefit 
from treatment while ensuring patient safety. 
Nivolumab, nivolumab-relatlimab or nivolumab and ipilimumab administration may be delayed for the 
following reasons: 
• 
Grade ≥2 non-skin, drug-related AEs with the following exceptions: 
• 
Grade 2 drug-related fatigue or laboratory abnormalities do not require a treatment delay 
• 
Any Grade 3 skin, drug-related AE 
• 
Any Grade 3 drug-related laboratory abnormalities with the following exceptions for lymphopenia, 
leukopenia, AST, ALT or total bilirubin 
• 
Grade 3 lymphopenia or leukopenia does not require dose delay 
• 
If a patient has a baseline AST, ALT or total bilirubin that is within normal limits, delay dosing for 
drug-related Grade ≥2 toxicity 
• 
If a patient has a baseline AST, ALT or total bilirubin that is within the Grade 1 toxicity range, delay 
dosing for drug-related Grade ≥3 toxicity 
• 
Any AE, laboratory abnormalities, or intercurrent illness, which in the judgment of the Investigator 
warrants delaying the dose of study drug. 
• 
All troponin elevations (including asymptomatic elevations) will require the participant to undergo a 
cardiac evaluation (via prompt cardiology consultation) and a confirmatory repeat evaluation within 24 
hours. If troponin elevation is not confirmed within 24 hours in an asymptomatic participant, a dose 
delay for the next dose may not be required provided that the cardiac evaluation is completed and and 
based on the Investigator’s judgment to proceed with treatment. 
• 
Otherwise, if the troponin elevation is confirmed, dosing may only resume when the AE resolves to 
baseline, and permanent discontinuation of treatment should be considered.  
 
7.10 Management guidelines for immune related adverse events 
Immuno-oncology (I-O) agents are associated with AEs that can differ in severity and duration than AEs 
caused by other therapeutic classes. Nivolumab, ipilimumab, and relatlimab are considered immuno-oncology 
agents in this protocol. Management algorithms have been developed to assist investigators in assessing and 
managing the following groups of AEs: Gastrointestinal, Renal, Pulmonary, Hepatic, Endocrinopathies, Skin, 
and Neurological. Early recognition and intervention are recommended according to the management 
algorithms; and in addition include ophthalmologic evaluations for any visual symptoms in order to evaluate for 
immune checkpoint-inhibitor related uveitis. 
For patients expected who require more than 4 weeks of corticosteroids or other immunosuppressants to 
manage an AE, consider the following recommendations. 

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• 
Antimicrobial/antifungal prophylaxis per institutional guidelines to prevent opportunistic infections 
such as Pneumocystis jiroveci and fungal infections. 
• 
Early consultation with an infectious disease specialist should be considered. 
• 
Depending on the presentation, consultation with a pulmonologist for bronchoscopy or a 
gastroenterologist for endoscopy may also be appropriate. 
• 
In patients who develop recurrent AEs in the setting of ongoing or prior immunosuppressant use, an 
opportunistic infection should be considered in the differential diagnosis. 
 
 
 

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7.11  Immune-Mediated Myocarditis  
Immune checkpoint inhibitors can cause immune-mediated myocarditis, which is defined as requiring use of 
steroids and no clear alternate etiology. The diagnosis of immune-mediated myocarditis requires a high index 
of suspicion. Patients with cardiac or cardio-pulmonary symptoms should be assessed for potential 
myocarditis. If myocarditis is suspected based on troponin elevation or clinical symptoms, withhold further 
therapy,confirm th elevation within 24 hours, arrange for a prompt cardiology followup and consider  promptly 
initiating high dose steroids (prednisone or methylprednisolone 1 to 2 mg/kg/day). If clinically confirmed, 
permanently discontinue nivolumab-relatlimab and HCQ for Grade 2-4 myocarditis. 
 
7.12 Hydroxychloroquine Dose Reduction 
Any AE of ≥ Grade 3 and attributed as possibly, probably or definitely related solely to HCQ will result in the dose 
being held until the AE has resolved to ≤ grade 1 or baseline while nivolumab, nivolumab and ipilimumab, or 
nivlumab-relatlimab dosing may continue uninterrupted. If the AE resolves, reinstitution of treatment can occur 
but at a reduced dose as described in Table 5. If the AE recurs at the reduced dose, treatment will be held until 
the AE has resolved to ≤ grade 1 and when resolved treatment can be reinstituted at the next lower dose level. 
No more than 2 dose reductions are allowed during the maintenance cycles. 
Table 5: Hydroxychloroquine Dose Reduction Schema 
Dose Level Dose mg/day 
First dose reduction  
Second Dose reduction  
 
• If no improvement, consider additional 
immunosuppression 
• Upon recovery, taper steroids over at least 1 
month with close monitoring of troponin and 
BNP as well as for new symptoms 
• Repeat cardiac MRI for post treatment 
assessments and cardiology follow-up 
• If worsens, intensify treatment according to 
grade 
• Upon recovery, taper steroids over at least 1 
month with close monitoring of troponin and 
BNP as well as for new symptoms 
• Repeat cardiac MRI for post treatment 
assessment and cardiology follow-up 
• Retreatment may be considered after recovery 
and completion of steroid taper 
• Monitor troponin until recovery 
 
• Resume I-O therapy upon recovery 
Follow-up 
• Permanently discontinue I-O therapy 
• Hospitalize to intensive cardiac monitoring 
• Cardiac evaluation to include: 
o 
Troponin and BNP monitoring 
o 
ECG  continuous cardiac monitoring 
o 
Echocardiogram 
o 
Cardiac MRI 
o 
Myocardial biopsy if feasible 
• Immediate initiation of 2 mg/kg/day methylprednisolone IV or 1 g IV 
bolus 
• Consider adding a second immunosuppressive agent 
Additionally, for life threatening myocarditis: 
• Hospitalize/transfer to institution with expertise in intensive cardiac 
monitoring 
• Consider ATG as second agent given its immediate effect 
Severe with symptoms at rest or 
with minimal activity or exertion; 
intervention indicated 
Life-threatening consequences; 
urgent intervention indicated (e.g., 
continuous IV therapy or 
mechanical hemodynamic support) 
RELATLIMAB MYOCARDITIS ADVERSE EVENT MANAGEMENT ALGORITHM 
For the protocols under CTCAE version 5.0. Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (eg, prednisone) at start of tapering or earlier, once 
sustained clinical improvement is observed. Lower bioavailability of oral corticosteroids should be taken into account when switching to the equivalent dose of oral corticosteroids. 
Prophylactic antibiotics should be considered in the setting of ongoing immunosuppression. 
ATG = anti-thymocyte globulin; BNP = B-type natriuretic peptide; ECG = electrocardiogram; IV = intravenous; MRI = magnetic resonance imaging 
Myocarditis 
 
Asymptomatic 
Troponin Elevation 
 
 
 
 
Symptoms with mild to 
moderate activity or exertion 
Management 
• Delay I-O therapy 
• Repeat and monitor troponin 
• Consider cardiology consultation with diagnostic workup; ECG, 
echocardiogram, cardiac MRI per recommendation of cardiologist 
• Consider corticosteroids per treating physician 
• Delay I-O therapy; hospitalization with cardiac monitoring 
• Urgent cardiology consultation for evaluation and management 
o 
Troponin and BNP 
o 
ECG  continuous cardiac monitoring 
o 
Echocardiogram 
o 
Cardiac MRI 
• Prompt initiation of 2 mg/kg/day methylprednisolone IV or 
equivalent 

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1 
400 mg twice daily 
400mg am and 200mg pm 
200 mg in am and pm 
2 
600 mg twice daily 
600mg am and 400mg pm 
400 mg in am and pm* 
1.5 
400 mg am/ 600 mg pm 
400mg twice daily 
200 mg in am and pm 
 
• 
A third dose reduction by 50% of the dose may be pursued in patients deemed to be deriving clinical 
benefit from the combination to HCQ 200 mg in am and pm at the discretion of the treating physician. 
Toxicities that may be attributable to HCQ include: nausea, anorexia, vomiting, constipation, diarrhea, rash, and 
visual field deficit.  If any of these AEs occur at grade ≤ 2, HCQ may be continued and the AE managed with 
supportive care. For any AE with a grade ≥ 3, HCQ dose will be held until the toxicity resolves to ≤ grade 2, after 
which HCQ may be restarted at a reduced dose as described in table 5.  With particular regard to visual field 
deficits patients should be cautioned to report any visual symptoms, particularly difficulty seeing entire words or 
faces, intolerance to glare, decreased night vision, or loss of peripheral vision. These symptoms of retinal 
toxicity or subclinical evidence of retinal toxicity on eye exam should prompt drug discontinuation and 
ophthalmologic evaluation. Patients who discontinue HCQ permanently may stay on nivolumab and stay on 
study at the discretion of the PI. 
7.13 Dose reductions for toxicities that overlap nivolumab, ipilimumab and HCQ 
Colitis, rash and fatigue can be seen with nivolumab, ipilimumab, and nivolumab-relatlimab and HCQ making it 
difficult to assign association with three drugs combined.  For rash and fatigue, HCQ can be attempted to be 
dose modified first before nivolumab and ipilimumab, or nivolumab-relatlimab. However, given the potential life 
threatening nature of colitis, all three drug should be stopped for Grade 3 diarrhea or grade 2 diarrhea with other 
associated symptoms (abdominal pain, fever) that is clearly not responsive to conservative diarrhea prevention 
measures. 
8.0 CORRELATIVE STUDIES 
8.1 Estimated number of subjects 
Maximum 70 subjects (combined enrollment in Phase 1a Nivolumab + HCQ dose escalation, Phase 2 
nivolumab + HCQ, Phase 2 nivolumab-relatlimab + HCQ, and Phase 1b nivolumab/ipilimumab + HCQ).  
Currently correlative studies will only be done in anti-PD-1 refractory patients due to funding constraints . 
However, tissue and blood will be collected on Phase 2b Cohort (PD-1 naïve) patients as well. 
 
8.2 Serial Tumor Biopsies and analysis 
 
Collection of Serial Tumor Biopsies 
The study will include optional serial tumor biopsies in any patient deemed appropriate for serial biopsy by the 
physician. If the tumor is not accessible by ultrasound guided biopsy or in office procedures, the patient may be 
deemed not appropriate for biopsy. Easily accessible lesions that do not require radiological guidance will be 
conducted either by the medical oncologists, surgical oncologist, dermatologists or dermatological surgeons. 
The procedures involved include 4 mm punch biopsies, excisional biopsies, excisions of large tumors in the 
operating room, and core needle biopsies. Lesions that are not easily accessible can be biopsied with the 
assistance of ultrasound guidance. This will involve core needle biopsies conducted by a radiologist.  In all 
cases patients will be consented separately for the surgical procedure and provide their written informed 
consent. Tumor biopsy material will be divided on ice by the operator and fixed in 
1) formalin 
2) snap frozen 
3) At the HUP main PENN site, fresh tumor tissue will be processed for flow cytomtery to assess TIL 
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Timepoints for serial tumor biopsies: 
1) baseline 
2) 12+/- 2 days after IO therapy + HCQ is started 
3) at progression 
We anticipate 20 patients out of 51 anti PD-1 refractory patients will be amenable to biopsy and with an 
average of 2.5 biopsies per patient, we anticipate a total of 50 biopsies. We will budget 50 radiology-assisted 
biopsies. 
8.3 Immunophenotyping on serially collected blood and tumor tissue 
Checkpoint blockade results in a change in the subsets of tumor infiltrating lymphocytes, and response to anti-
PD1 therapy is correlated with an increase in CD8 T cell infiltrate and PDL-1 expression in the tumor.  There is 
also increasing data that suggest that HCQ modulates immunosuppressive myeloid populations in the tumor 
microenvironment. We have in vitro data that M2 (immunosuppressive) to M1 (anti-tumor) macrophage 
phenotype switching occurs with HCQ treatment. We have in vivo data that HCQ + PD1 results in a significant 
reduction in polymorphonuclear myeloid derived suppressor cells (PMN-MDSC). 
Dr. Alexander Huang at the University of Pennsylvania has pioneered immunophenotyping of peripheral blood 
(measuring re-invigoration of exhausted phenotype CD8 T cells) in melanoma patients treated with anti-PD1 
antibody pembrolizumab (Huang et al. Nature 2017). It is currently unknown whether a similar immunological 
response can be identified in patients on IO and autophagy targeted therapies. In patients undergoing serial 
biopsy, we will collect paired blood (Peripheral Blood Mononuclear Cells (PBMC)) and tumor specimens at pre-
treatment, and 12 days , 4 weeks, 8 weeks, 12 weeks and 12 months on treatment (for nivo + HCQ or 
nivolumab + relatlimab + HCQ), or 3 weeks 6 weeks, 9 weeks, 12 weeks, 12 months for ipi + nivo.  We will 
also use the freshly collected tumor piece to isolate immune infiltrates for 4-8 color multicolor 
immunofluorescence to interrogate the components of the immune system (CD8, Tregs, myeloid) in the tumor 
microenvironment and changes after nivolumab and HCQ or nivolumab and ipilimumab and HCQ. 
Budget. We will focus immune analysis on patients who also undergo serial tumor biopsy and we anticipate 
there will be 20 patients who undergo serial tumor biopsy (pre and post). For these 20 patients, we will collect 
a peripheral blood at 4 time points, pre-treatment and weeks 2, 4, 8 post treatment for a total of 80 peripheral 
blood samples. In addition, CD45 cells will be isolated from 50 tumors. Funds are requested to purchase 
flurochrome tagged antibodies for flow cytometry analysis. Twenty-three separate antibodies to different 
targets will be purchased. There will be total of 130 samples (see above) each of which will be analyzed with 
23 antibodies for a total of 2070 stains. 
 
8.4 Tracking tumor specific T cells using combinatorial tetramer 
We will test whether blockade of PD-1 and autophagy pathways alter the number, phenotype, and function of 
tumor-specific and viral-specific T cells in the tumor.  We have recently established the combinatorial tetramer 
panel where we can track 10 antigen-specific T cell populations using 4 flow cytometry channels (Fig. 4). We 
will use this platform to screen for tumor and viral-specific T cells and track the kinetics of the immune 
response to PD1+autophagy blockade, and interrogate transcriptional profile, inhibitory expression, and 
function of tumor-specific T cells.  On-target effects (tumor-specific changes), and off-target effects (viral-
specific changes) can then be associated with clinical outcomes such as clinical response and irAEs. 
 
8.5 Immunohistochemistry for markers of tumor immunity in serial biopsies 
In parallel immune markers well be measured by IHC in serial tumor biopsies including MART1 (melanoma), 
CD8+ (cytotoxic T cells), F4/80 (TAM), Ly6G/C (MDSC). Using multiplexed IHC we will be able to determine if 
the addition of HCQ reduces PMN-MDSC, or converts M2 to M1 TAMs, and results in increased infiltration of T 
cells into the tumor. 

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8.6 Nanostring on serially collected tumor tissue 
While informative, there are limited antibodies to a limited amount of targets for a comprehensive evaluation of 
the tumor microenvironment by immunofluorescence. It has been demonstrated that gene expression analysis 
is highly effective in characterizing the immune contexture of tumors using validated immune signatures. We 
will nanostring platform, which measures expression patterns for focused number of genes in paraffin-
embedded tumors, before and after treatment, to compare changes in immune and tumor signatures after 
treatment with nivolumab, or nivolumab and ipilimumab, plus HCQ. 33 
 
8.7 FFPE archival tumor tissue for immunohistochemistry 
For all anti PD-1 refractory patients (51), we plan to obtain 20 unstained slides from archival FFPE blocks of 
ideally the latest metastatic lesion that was biopsied will be obtained. IHC assays for correlative autophagy 
biomarkers:  We have identified a 2-gene signature involving Aldehyde dehydrogenase and helicase like 
transcription factor (HLTF) that predicts antitumor sensitivity of cancer cell lines to HCQ.  We have developed 
IHC protocols for these genes. We hypothesize the patterns of expression of these genes previously identified 
in HCQ-S and HCQ-R cancer cell lines (Piao Autophagy 2017) will correlate with PFS. In addition, the following 
proteins will be measured by IHC: PDL1, HLTF, ALDH1A1, PPT1. Additional biomarkers and Immune cell 
staining will be conducted as the concept is developed further. 
 

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9.0 STUDY CALENDAR (TABLE 6) 
(SEE APPENDIX B FOR RECOMMENDED VISIT BY VISIT ASSESSMENTS) 
 
 
 
During Protocol Therapy 
Post-
Treatment 
 
Baseline / 
Pre-RXa 
Day 12 
+/- 2 days 
Each cycle1 
End of 
protocol 
treatment 
(any reason) 
Disease 
Progression 
Follow-up*** 
 
History and physical 
examination 
X 
 
X 
 
 
Safety follow-
up 100 days 
after last dose 
of study drug 
Vital Signs (particularly BP) 
X 
 
X 
 
 
 
EKG2  
X 
 
X 
 
 
 
Brain CT or MRI 
X 
 
Every 3-6 cycles if brain 
metastases are present 
on baseline scan 
 
 
As clinically 
indicated 
Chest CT3 
X 
 
Every 3 cycles and as 
clinically indicated 
 
 
As clinically 
indicated 
Abdomen/pelvis CT or MRI3 
X 
 
Every 3 cycles and as 
clinically indicated 
Confirmatory scans at 
least 4 weeks following 
initial documentation of 
objective response 
 
 
As clinically 
indicated 
CBC with differential 
X 
 
X 
X 
 
 
Troponin Sodium, potassium, 
BUN, serum creatinine, 
glucose, SGOT (AST), SGPT 
(ALT), total bilirubin, alkaline 
phosphatase, LDH, albumin; 
TSH  
X 
 
X 
X 
 
 

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During Protocol Therapy 
Post-
Treatment 
 
Baseline / 
Pre-RXa 
Day 12 
+/- 2 days 
Each cycle1 
End of 
protocol 
treatment 
(any reason) 
Disease 
Progression 
Follow-up*** 
PT/PTT 
X 
 
 
 
 
 
Serum ΒHCG4 
X 
 
 
 
 
 
Urine pregnancy test 
X (within 24 
hours of 
starting) 
 
Before each cycle 
(within 3 days of dosing)  
 
 
 
 
 
 
 
 
 
 
TREATMENT 
 
 
 
 
 
 
 
 
 
 
 
 
 
Phase 1a, 2a, 2b, 2c 
(HCQ+nivolumab) 
 
 
 
 
 
 
Nivolumab 
or 
nivolumab-
relatlimab 
 
 
X 
every 4 weeks 
 
 
 
Hydroxychloroquine 
 
 
Twice every day 
 
 
 
 
 
 
 
 
 
 
Phase 1b 
(HCQ+nivolumab+ipilimumab) 
 
 
 
 
 
 
Nivolumab 
 
 
X 
every 3 weeks x4 cycles 
 
followed by Maintenance 
every 4 weeks (starting 
6 weeks after last dose 
of ipi/nivo) 
 
 
 
Ipilimumab 
 
 
X 
every 3 weeks x4 cycles 
 
 
 

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During Protocol Therapy 
Post-
Treatment 
 
Baseline / 
Pre-RXa 
Day 12 
+/- 2 days 
Each cycle1 
End of 
protocol 
treatment 
(any reason) 
Disease 
Progression 
Follow-up*** 
Hydroxychloroquine 
 
 
Twice every day 
 
 
 
CORRELATIVE STUDIES8 
 
 
 
 
 
 
Research tumor biopsy in 
biopsiable patients5  
X 
X 
 
 
X 
 
Plasma collection for autophagy 
biomarker (1 purple top) 
X for all 
patients 
 
X day 12 
only for 
biopsy pts 
X6 for all patients 
X 
 
 
PBMC collection (for biopsy 
patients only)7  
X 
X 
X (4, 8, 12 weeks only 
for nivo + HCQ or nivo-
rela + HCQ, or 3, 6, 9, 
12 weeks for ipi + nivo). 
All patients with serial 
PBMC collection will 
have one additional 
draw at 12 months 
 
 
 
Archival Tumor tissue  
X9 
 
 
 
 
 
 
Standard of care *To account for major holidays and unforeseen events such as inclement weather, scheduled visits and testing can occur +/- 1 week, 
as long as there is documentation as to why they did not occur according to intended schedule 
***  Follow-up: all subjects will be evaluated for at least 100 days after discontinuation of study treatment and will be followed for a minimum of one 
year from the start of treatment, unless disease progression is reported prior to one year.  One-year survival information will be extracted from the 
medical record or by phone call for subjects who have discontinued protocol treatment and completed the 100 day follow-up evaluations prior to one 
year from the start of treatment. 
a- eligibility labs need to be completed within 14 days before start of treatment. Eligibility/baseline scans need to be within 28 days of starting treatment. 
 

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NOTE: All visceral areas originally involved by tumor should be re-imaged every 3 cycles. In the case of bone scan, if negative at baseline, it may be 
deferred until the development of bone pain or a newly increased alkaline phosphatase. 
1. A cycle constitutes 28 days or 4 weeks of nivolumab + HCQ  or nivolumab-relatlimab+HCQ therapy and 21 days for the first 4 combined treatments 
with nivolumab + ipilimumab and HCQ, and 28 days thereafter for maintenance nivolumab and HCQ.  Monthly tests and procedures should be 
done on Day 1 of each cycle +/- 1 week. Screening labs can be used for cycle 1 day 1 if obtained within 14 days.  
2. If HCQ is discontinued, EKGs are only required if clinically indicated.  
3. Standard of care imaging will be utilized for tumor measurements while on study. The preferred type of imaging is high resolution CT scans of the 
chest abdomen and pelvis with contrast. In cases of elevated creatinine or anaphylactic reaction to contrast dye, CT scans without contrast or 
MRI with gadolinium may be substituted for the abdomen and non-contrast CT for the chest. PET/CT scans may not be substituted unless the CT 
portion of the PET/CT is a high-resolution scan with contrast. Scans will be obtained every 12 weeks +/- 1 week.  Ipi/nivo patients would be getting 
first ontreatment scans after 4 cycles and then after every 3 cycles of maintenance nivolumab.  
4. For women of childbearing potential. Serum HCG within 28 days of starting treatment and urine pregnancy test within 24 hours of starting should 
be obtained. 
5. Tumor biopsies will be obtained on patients that have easily accessible lesions, amenable to at least one biopsy, and whose biopsiable tumor is 
not the only RECIST measurable disease. Window for tumor biopsy will be +/- 2 days. 
6. Plasma will be collected every cycle for the first 3 months in all patients. 
7. Please refer to Section 8.3 for details of sample collection. 
8. Patients are asked to continue drug treatment as prescribed and not hold any drug doses. 
9. Archival tumor tissue can be obtained at any point during study enrollment and treatment timeline. 
 

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10.0 
MEASUREMENT OF EFFECT 
Patients will be evaluable for response if they have completed 75% of their expected dose of HCQ for 4 weeks 
of combination therapy with immune checkpoint inhibitor. These restrictions do not apply to patients that had a 
DLT (see Section 5.2 for rules regarding evaluability for patients experiencing DLT).  For the purposes of this 
study, patients should be re-evaluated for response every 3 cycles (12 weeks).  In addition to a baseline scan, 
confirmatory scans should also be obtained (not less than 4) weeks following initial documentation of objective 
response. 
Response and progression will be evaluated in this study using the new international criteria proposed by the 
revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1) [Eur J Ca 45:228-247, 
2009]. Investigator assessment of RECIST response at each site will be used to make treatment decisions 
in real time. 
10.1 Definitions 
Evaluable for toxicity.  All patients will be evaluable for toxicity from the time of their first treatment with combined 
treatment with nivolumab and HCQ. 
Evaluable for objective response.  Only those patients who have measurable disease present at baseline, have 
received at least one cycle of therapy, if they have completed 75% of their expected dose of HCQ for 4 weeks 
of combination therapy with nivolumab, and have had their disease re-evaluated will be considered evaluable 
for response.  These patients will have their response classified according to the definitions stated below.  (Note:  
Patients who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.) 
10.2 Disease Parameters 
Measurable disease.  Measurable lesions are defined as those that can be accurately measured in at least one 
dimension (longest diameter to be recorded) as >20 mm by chest x-ray, as >10 mm with CT scan, or >10 mm 
with calipers by clinical exam.  All tumor measurements must be recorded in millimeters (or decimal fractions of 
centimeters). 
Note:  Tumor lesions that are situated in a previously irradiated area are not considered measurable. 
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a lymph node must be >15 
mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 
mm).  At baseline and in follow-up, only the short axis will be measured and followed. 
Non-measurable disease.  All other lesions (or sites of disease), including small lesions (longest diameter <10 
mm or pathological lymph nodes with ≥ 10 to <15 mm short axis), are considered non-measurable disease.  
Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, 
inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-
measurable. 
Note:  Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as 
malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 
 ‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable lesions, if they meet 
the definition of measurability described above. However, if non-cystic lesions are present in the same patient, 
these are preferred for selection as target lesions. 
Target lesions.  All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, 
representative of all involved organs, should be identified as target lesions and recorded and measured at 
baseline.  Target lesions should be selected based on their size (lesions with the longest diameter), be 
representative of all involved organs, but in addition should be those that lend themselves to reproducible 

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repeated measurements.  It may be the case that, on occasion, the largest lesion does not lend itself to 
reproducible measurement in which circumstance the next largest lesion, which can be measured reproducibly, 
should be selected.  A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all 
target lesions will be calculated and reported as the baseline sum diameters.  If lymph nodes are to be included 
in the sum, then only the short axis is added into the sum.  The baseline sum diameters will be used as reference 
to further characterize any objective tumor regression in the measurable dimension of the disease. 
Non-target lesions.  All other lesions (or sites of disease) including any measurable lesions over and above the 5 
target lesions should be identified as non-target lesions and should also be recorded at baseline.  Measurements 
of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each 
should be noted throughout follow-up. 
10.3 Methods for Evaluation of Measurable Disease 
All measurements should be taken and recorded in metric notation using a ruler or calipers.  All baseline 
evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 
weeks before the beginning of the treatment. 
Clinical lesions.  Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules 
and palpable lymph nodes) and ³10 mm diameter as assessed using calipers (e.g., skin nodules).  In the case 
of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is 
recommended. 
Chest x-ray.  Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and 
surrounded by aerated lung.  However, CT is preferable. 
Conventional CT and MRI.  This guideline has defined measurability of lesions on CT scan based on the 
assumption that CT slice thickness is 5 mm or less.  If CT scans have slice thickness greater than 5 mm, the 
minimum size for a measurable lesion should be twice the slice thickness.  MRI is also acceptable in certain 
situations (e.g. for body scans). 
10.4 Response Criteria 
10.4.1 Evaluation of Target Lesions 
 
Complete Response (CR): 
Disappearance of all target lesions.  Any pathological lymph nodes (whether target 
or non-target) must have reduction in short axis to <10 mm. 
Partial Response (PR): 
At least a 30% decrease in the sum of the diameters of target lesions, taking as 
reference the baseline sum diameters 
Confirmed Partial Response (cPR): 2 scans in a row separate by 4 weeks that show a partial response. 
Progressive Disease (PD): 
At least a 20% increase in the sum of the diameters of target lesions, taking as 
reference the smallest sum on study (this includes the baseline sum if that is the smallest on study).  In addition 
to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm.  (Note:  
the appearance of one or more new lesions is also considered progressions). 
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking 
as reference the smallest sum diameters while on study 

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10.4.2 Evaluation of Non-Target Lesions 
 
Complete Response (CR): 
Disappearance of all non-target lesions and normalization of tumor marker level.  
All lymph nodes must be non-pathological in size (<10 mm short axis) 
Note:  If tumor markers are initially above the upper normal limit, they must normalize for a patient to be 
considered in complete clinical response. 
Non-CR/Non-PD: 
Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level 
above the normal limits 
Progressive Disease (PD): 
Appearance of one or more new lesions and/or unequivocal progression of existing 
non-target lesions.  Unequivocal progression should not normally trump target lesion status.  It must be 
representative of overall disease status change, not a single lesion increase. 
Although a clear progression of “non-target” lesions only is exceptional, the opinion of the treating physician 
should prevail in such circumstances, and the progression status should be confirmed at a later time by the 
review panel (or Principal Investigator). 
 
10.4.3 Evaluation of Best Overall Response 
 
The best overall response is the best response recorded from the start of the treatment until disease 
progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since 
the treatment started).  The patient's best response assignment will depend on the achievement of both 
measurement and confirmation criteria. 
Table 7. Determination of Best Overall Response 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response 
when Confirmation is 
Required* 
CR 
CR 
No 
CR 
>4 wks. Confirmation** 
CR 
Non-CR / Non-PD 
No 
PR 
 
>4 wks. Confirmation** 
CR 
Not evaluated 
No 
PR 
PR 
Non-CR / Non-PD 
/ not evaluated 
No 
PR 
SD 
Non-CR / Non-
PD/not evaluated 
No 
SD 
Documented at least 
once >4 wks. from 
baseline** 
PD 
Any 
Yes or No 
PD 
 
no prior SD, PR or CR 
Any 
PD*** 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
*      See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
**    Only for non-randomized trials with response as primary endpoint. 
***  In exceptional circumstances, unequivocal progression in non-target lesions may be 
accepted as disease progression. 
Note: Patients with a global deterioration of health status requiring discontinuation of treatment 
without objective evidence of disease progression at that time should be reported as 

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10.5 
Duration of Response 
Duration of overall response:  The duration of overall response is measured from the time measurement 
criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive 
disease is objectively documented (taking as reference for progressive disease the smallest measurements 
recorded since the treatment started). 
The duration of overall CR is measured from the time measurement criteria are first met for CR until the first 
date that progressive disease is objectively documented. 
Duration of stable disease:  Stable disease is measured from the start of the treatment until the criteria for 
progression are met, taking as reference the smallest measurements recorded since the treatment started, 
including the baseline measurements. 
10.6 Progression-Free Survival 
PFS is defined as the duration of time from start of treatment to time of first progression, death due to any 
cause or last patient contact alive and progression-free. In patients treated beyond progression, time to first 
progression (isolated) and second progression (multifocal) will be recorded. 
10.7 Off Treatment/Off Study 
Each subject has the right to withdraw from the study at any time without prejudice.  The investigator may 
discontinue any subject’s participation for any reason, including adverse event or failure to comply with the 
protocol.  Should a subject withdraw from the study, the reason(s) must be stated on the case report form, and 
a final evaluation of the subject should be performed. Reasons for withdrawal include the following: 
Progression of Disease:  Remove patient from protocol therapy at the time progressive disease is documented. 
Extraordinary Medical Circumstance: If at any time the treating physician feels constraints of this protocol are 
detrimental to the patient’s health remove the patient from protocol therapy. 
Patient’s refusal to continue treatment: In this event, document the reason(s) for withdrawal. 
Failure to comply with protocol (as judged by the investigator such as compliance below 80%, failure to 
maintain appointments, etc.). 
Delay in treatment > 28 days due to toxicity 
11.0 
ADVERSE EVENTS AND REPORTING 
The timely reporting of adverse events (including toxic deaths) is required by the Food and Drug 
Administration.  The reporting of toxicities is part of the data reporting for this study.  The investigator is 
responsible for ensuring that all adverse events (AEs) and significant adverse events (SAEs) that are observed 
or reported during the study are collected and reported to the FDA, appropriate IRB(s), in accordance with 
CFR 312.32 (IND Safety Reports). 
11.1 Adverse Events 
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in severity during 
the course of the study that does not necessarily have a causal relationship with this treatment.  Intercurrent 
illnesses or injuries should be regarded as adverse events.  Abnormal results of diagnostic procedures are 
considered to be adverse events if the abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
“symptomatic deterioration.”  Every effort should be made to document the objective 
progression even after discontinuation of treatment. 

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• leads to additional treatment or to further diagnostic tests is considered by the investigator to be of 
clinical significance 
A non-serious adverse event is an AE not classified as serious. 
 
Note: Although not always adverse events by regulatory definition, the following events associated with a BMS 
product must be reported. 
• 
Exposure (to fetus) during pregnancy, exposure (to infant) during lactation, and paternal exposure 
• 
Overdose 
• 
Lack of efficacy 
• 
Abuse 
• 
Misuse 
• 
Off-label use 
• 
Occupational exposure 
• 
Medication error and potential medication error 
• 
Suspected transmission of an infectious agent, e.g., any organism, virus or infectious particle 
pathogenic or non-pathogenic, via the medicinal product. 
 
Adverse Event Reporting Period. 
The study period during which adverse events must be reported is defined as the period from the initiation of 
the first study treatment to 100 days following the last administration of study treatment. 
Post-study Adverse Event. 
All unresolved adverse events should be followed by the investigator until the events are resolved, the patient 
is lost to follow-up, or the adverse event is otherwise explained. At the last scheduled visit, the investigator 
should instruct each patient to report any subsequent event(s) that the patient, or the patient’s personal 
physician, believes might reasonably be related to participation in this study. 
Abnormal Laboratory Values. 
A clinical laboratory abnormality should be documented as an adverse event if the abnormality is of a degree, 
typically at least grade 2 and not present as grade 1 or better at baseline, that requires active management; 
e.g. change of dose, discontinuation of the drug, more frequent follow-up assessments, further diagnostic 
investigation. 
11.2 Recording of Adverse Events 
At each contact with the subject, the investigator must seek information on adverse events by specific 
questioning and, as appropriate, by examination.  Information on all adverse events should be recorded 
immediately in the source document, and also in the appropriate adverse event module of the case report form 
(CRF).  All clearly related signs, symptoms, and abnormal diagnostic procedures results should be recorded in 
the source document, though should be grouped under one diagnosis. 
All adverse events occurring during the study period must be recorded. Adverse events will be measured and 
graded in accordance with the document entitled “Common Terminology Criteria for Adverse Events 
(CTCAE)”, NCI version 5.0 issued by the US Department of Health and Human Services. The clinical course of 
each event should be followed until resolution, stabilization, or until it has been determined that the study 
treatment or participation is not the cause.  Serious adverse events that are still ongoing at the end of the study 
period must be followed up to determine the final outcome.  Any serious adverse event that occurs after the 
study period and is considered to be at least possibly related to the study treatment or study participation 
should be recorded and reported immediately. 

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11.2.1 Serious Adverse Events 
 
Adverse events are classified as serious or non-serious. 
A serious adverse event is any AE that is: 
• fatal 
• life-threatening 
• requires or prolongs hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• Suspected transmission of an infectious agent (e.g., pathogenic or nonpathogenic) via the study drug is 
an SAE. 
• an important medical event 
Important medical events are those that may not be immediately life threatening, but are clearly of major 
clinical significance.  They may jeopardize the subject, and may require intervention to prevent one of the other 
serious outcomes noted above.  For example, drug overdose or abuse, a seizure that did not result in in-
patient hospitalization, or intensive treatment of bronchospasm in an emergency department would typically be 
considered serious. 
ADVERSE EVENT COLLECTION AND REPORTING INFORMATION: 
 
• 
All Serious Adverse Events (SAEs) that occur following the subject’s written consent to participate in 
the study through 100 days of discontinuation of dosing must be reported to BMS Worldwide Safety, 
whether related or not related to study drug. If applicable, SAEs must be collected that relate to any 
later protocol-specified procedure (e.g., a follow-up skin biopsy). 
• 
Following the subject’s written consent to participate in the study, all SAEs, whether related or not 
related to study drug, are collected, including those thought to be associated with protocol-specified 
procedures. The investigator should report any SAE occurring after these aforementioned time periods, 
which is believed to be related to study drug or protocol-specified procedure. 
• 
An SAE report should be completed for any event where doubt exists regarding its seriousness; 
• 
If the investigator believes that an SAE is not related to study drug, but is potentially related to the 
conditions of the study (such as withdrawal of previous therapy or a complication of a study procedure), 
the relationship should be specified in the narrative section of the SAE Report Form. 
 
An appropriate SAE form (e.g.  ex-US = CIOMS form or USA = MedWatch form) should be used to report 
SAEs to BMS.  If you prefer to use your own Institutional form, it must be reviewed by the BMS Protocol 
Manager prior to study initiation to ensure that at a minimum all of the data elements on the CIOMS form are 
present.  Note:  Please include the BMS Protocol number on the SAE form or on the cover sheet with the SAE 
form transmission. 
o 
The CIOMS form is available at: http://www.cioms.ch/index.php/cioms-form-i 
o 
The MedWatch form is available at: MedWatch 3500 Form 
• 
For studies with long-term follow-up periods in which safety data are being reported, include the timing 
of SAE collection. 
• 
The Sponsor will reconcile the clinical database AE cases (case level only) transmitted to BMS Global 
Pharmacovigilance (Worldwide.Safety@bms.com). 

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• 
The Investigator will request from BMS GPV&E, aepbusinessprocess@bms.com the SAE reconciliation 
report and include the BMS protocol number every 3 months and prior to data base lock or final data 
summary 
• 
GPV&E will send the investigator the report to verify and confirm all SAEs have been transmitted to 
BMS GPV&E. 
• 
The data elements listed on the GPV&E reconciliation report will be used for case identification 
purposes. If the Investigator determines a case was not transmitted to BMS GPV&E, the case should 
be sent immediately to BMS (Worldwide.Safety@bms.com). 
• 
In addition to the Sponsor Investigator’s responsibility to report events to their local HA, suspected 
serious adverse reactions (whether expected or unexpected) shall be reported by BMS to the relevant 
competent health authorities in all concerned countries according to local regulations (either as 
expedited and/or in aggregate reports). 
• 
In accordance with local regulations, BMS will notify sponsor investigators of all reported SAEs that are 
suspected (related to the investigational product) and unexpected (i.e., not previously described in the 
IB). An event meeting these criteria is termed a Suspected, Unexpected Serious Adverse Reaction 
(SUSAR). Sponsor investigator notification of these events will be in the form of either a SUSAR Report 
or a Semi-Annual SUSAR Report. 
o 
Other important findings which may be reported by BMS as an Expedited Safety Report (ESR) 
include: increased frequency of a clinically significant expected SAE, an SAE considered 
associated with study procedures that could modify the conduct of the study, lack of efficacy 
that poses significant hazard to study subjects, clinically significant safety finding from a 
nonclinical (e.g., animal) study, important safety recommendations from a study data 
monitoring committee, or sponsor or BMS decision to end or temporarily halt a clinical study for 
safety reasons. 
o 
Upon receiving an ESR from BMS, the investigator must review and retain the ESR with the IB. 
Where required by local regulations or when there is a central IRB for the study, the sponsor 
will submit the ESR to the appropriate IRB. The investigator and IRB will determine if the 
informed consent requires revision. The investigator should also comply with the IRB 
procedures for reporting any other safety information. 
 
SAEs, whether related or not related to study drug, and pregnancies must be reported to BMS within 24 hours \ 
1 Business Day of becoming aware of the event. SAEs must be recorded on either CIOMS, MedWatch, or 
approved site SAE form. 
Pregnancies must be reported and submitted to BMS.  BMS will perform due diligence follow-up using the 
BMS Pregnancy Form which the investigator must complete. 
SAE Email Address:  Worldwide.Safety@BMS.com 
SAE Facsimile Number:  +1 609-818-3804 
If only limited information is initially available, follow-up reports are required. (Note: Follow-up SAE reports 
should include the same investigator term(s) initially reported.) 
If an ongoing SAE changes in its intensity or relationship to study drug or if new information becomes 
available, a follow-up SAE report should be sent within 24 hours to BMS using the same procedure used for 
transmitting the initial SAE report. 
All SAEs should be followed to resolution or stabilization. 

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The causal relationship to study drug is determined by a physician and should be used to assess all adverse 
events (AE). The casual relationship can be one of the following: 
• 
Related: There is a reasonable causal relationship between study drug administration and the AE. 
• 
Not related: There is not a reasonable causal relationship between study drug administration and the 
AE. 
The term "reasonable causal relationship" means there is evidence to suggest a causal relationship. 
Adverse events can be spontaneously reported or elicited during open-ended questioning, examination, or 
evaluation of a subject. (In order to prevent reporting bias, subjects should not be questioned regarding the 
specific occurrence of one or more AEs.) 
 
All adverse events that do not meet any of the criteria for serious should be regarded as non-serious 
adverse events. 
NONSERIOUS ADVERSE EVENT 
 
• 
Non-serious Adverse Events (AE) are to be provided to BMS in aggregate via interim or final study 
reports as specified in the agreement or, if a regulatory requirement [e.g., IND US trial] as part of an 
annual reporting requirement. 
• 
Non-serious AE information should also be collected from the time of the subject’s written consent to 
participate in the study. 
 
Non-serious Adverse Event Collection and Reporting 
The collection of non-serious AE information should begin following the subject’s written consent to participate 
in the study. All non-serious adverse events (not only those deemed to be treatment-related) should be 
collected continuously during the treatment period and for a minimum of 100 days following the last dose of 
study treatment. 
Non-serious AEs should be followed to resolution or stabilization, or reported as SAEs if they become serious. 
Follow-up is also required for non-serious AEs that cause interruption or discontinuation of study drug and for 
those present at the end of study treatment as appropriate. 
 
Laboratory Test Abnormalities 
All laboratory test results captured as part of the study should be recorded following institutional procedures. 
Test results that constitute SAEs should be documented and reported to BMS as such. 
The following laboratory abnormalities should be documented and reported appropriately: 
• 
any laboratory test result that is clinically significant or meets the definition of an SAE 
• 
any laboratory abnormality that required the participant to have study drug discontinued or interrupted 
• 
any laboratory abnormality that required the subject to receive specific corrective therapy. 
It is expected that wherever possible, the clinical rather than laboratory term would be used by the reporting 
investigator (e.g., anemia versus low hemoglobin value). 
 

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Hospitalization, Prolonged Hospitalization or Surgery. 
Any adverse event that results in hospitalization or prolonged hospitalization should be documented and 
reported as a serious adverse event unless specifically instructed otherwise in this protocol. Any condition 
responsible for surgery should be documented as an adverse event if the condition meets the criteria for an 
adverse event. 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an adverse event 
in the following circumstances: 
• 
Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures for a preexisting 
condition. Surgery should not be reported as an outcome of an adverse event if the purpose of the 
surgery was elective or diagnostic and the outcome was uneventful. 
• 
Hospitalization or prolonged hospitalization required to allow efficacy measurement for the study. 
• 
Hospitalization or prolonged hospitalization for therapy of the target disease of the study, unless it is a 
worsening or increase in frequency of hospital admissions as judged by the clinical investigator. 
 
11.3 Assessment of Adverse Events 
All AEs and SAEs whether volunteered by the subject, discovered by study personnel during questioning, or 
detected through physical examination, laboratory test, or other means will be reported appropriately. Each 
reported AE or SAE will be described by its duration (i.e., start and end dates), regulatory seriousness criteria if 
applicable, suspected relationship to the study drug (see following guidance), and actions taken. To ensure 
consistency of AE and SAE causality assessments, only the PI or sub-investigators may grade AEs, and 
investigators should apply the following general guideline: 
11.3.1 Relationship to study drug: Yes 
 
There is a plausible temporal relationship between the onset of the AE and administration of the study drug, 
and the AE cannot be readily explained by the subject’s clinical state, intercurrent illness, or concomitant 
therapies; and/or the AE follows a known pattern of response to the study drug; and/or the AE abates or 
resolves upon discontinuation of the study drug or dose reduction and, if applicable, reappears upon re-
challenge. 
11.3.2 Relationship to study drug: No 
 
Evidence exists that the AE has an etiology other than the study drug (e.g., preexisting medical condition, 
underlying disease, intercurrent illness, or concomitant medication); and/or the AE has no plausible temporal 
relationship to study drug administration (e.g., cancer diagnosed 2 days after first dose of study drug). 
Expected adverse events are those adverse events that are listed or characterized in the Package Insert or 
current Investigator Brochure. 
Unexpected adverse events are those not listed in the Package Insert (P.I.) or current Investigator Brochure 
(I.B.) or not identified. This includes adverse events for which the specificity or severity is not consistent with 
the description in the P.I. or I.B. For example, under this definition, hepatic necrosis would be unexpected if the 
P.I. or I.B. only referred to elevated hepatic enzymes or hepatitis. 
11.3.3 Diagnosis vs. Signs and Symptoms 
 
If known at the time of reporting, a diagnosis should be reported rather than individual signs and symptoms 
(e.g., record only liver failure or hepatitis rather than jaundice, asterixis, and elevated transaminases). 
However, if a constellation of signs and/or symptoms cannot be medically characterized as a single diagnosis 

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or syndrome at the time of reporting, it is ok to report the information that is currently available. If a diagnosis is 
subsequently established, it should be reported as follow-up information. 
11.3.4 Deaths 
 
All deaths that occur during the protocol-specified AE reporting period (see Section 12.1), regardless of 
attribution, will be reported to the appropriate parties. When recording a death, the event or condition that 
caused or contributed to the fatal outcome should be reported as the single medical concept. If the cause of 
death is unknown and cannot be ascertained at the time of reporting, report “Unexplained Death”. 
11.3.5 Preexisting Medical Conditions 
 
A preexisting medical condition is one that is present at the start of the study. Such conditions should be 
reported as medical and surgical history. A preexisting medical condition should be re-assessed throughout the 
trial and reported as an AE or SAE only if the frequency, severity, or character of the condition worsens during 
the study. When reporting such events, it is important to convey the concept that the preexisting condition has 
changed by including applicable descriptors (e.g., “more frequent headaches”). 
11.3.6 Pregnancy 
 
If a female subject becomes pregnant while receiving investigational therapy or within 120 days after the last 
dose of study drug, the investigator must immediately notify Worldwide.Safety@BAMS.com of this event via 
the Pregnancy Surveillance From (provided from BMS upon request) in accordance with SAE reporting 
guidelines.  Follow-up to obtain the outcome of the pregnancy should also occur. Abortion, whether accidental, 
therapeutic, or spontaneous, should always be classified as serious, and expeditiously reported as an SAE. 
Similarly, details of the birth, and the presence or absence of any congenital anomaly/birth defect or maternal 
and/or newborn complications in a child born to a female subject exposed to the study drug should be reported 
as an SAE. Follow-up information regarding the course of the pregnancy, including perinatal and neonatal 
outcome and, where applicable, offspring information must be reported on the Pregnancy Surveillance Form 
[provided upon request from BMS]. In order for Sponsor or designee to collect any pregnancy surveillance 
information from the female partner must sign an informed consent form for disclosure of this information.  Any 
pregnancy that occurs in a female partner of a male study participant should be reported to BMS. Information 
on this pregnancy will be collected on the Pregnancy Surveillance Form. In order for Sponsor or designee to 
collect any pregnancy surveillance information from the female partner, the female partner must sign an 
informed consent form for disclosure of this information. 
 
11.4 UPENN Abramson Cancer Center (ACC)’s Data and Safety Monitoring Committee (DSMC) 
Notification by all Sites 
The Data Safety and Monitoring Committee (DSMC) of the University of Pennsylvania’s Abramson Cancer 
Center will monitor the data quality and adherence to safety rules. Additionally, the DSMC will review all 
safety/toxicity data for the trial and recommend trial suspension or termination as needed. Specific details of 
monitoring and audit frequency will be included in the monitoring plan, but will be at least every 6 months. 
Medical Monitor 
This study will be monitored in accordance with the DSMC monitoring plan. Dr. Noelle Frey, an independent 
clinician in the Department of Medicine, Division of Hematology Oncology, will serve as medical monitor and 
will consult on decisions made as a part of this trial as noted above. The medical monitor will perform a regular 
review and assessment of the number and type of serious adverse events (at minimum yearly, but may occur 
more frequently as needed). 

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It is the responsibility of the Principal Investigator to oversee the safety of the study. This safety monitoring will 
include careful assessment and appropriate reporting of adverse events, as noted above, as well as the 
construction and implementation of a site data and safety monitoring plan. 
 
Notifying the Data Safety Monitoring Committee (DSMC) for the following Adverse Events 
1. 
All grade 3 or higher events regardless of attribution or expectedness within 10 business days of 
knowledge. 
2. 
All unexpected deaths within two business days of knowledge. 
3. 
All other deaths within 30 days of knowledge. Deaths of subjects greater than 90 days from the last 
study treatment/intervention are not reportable. 
AEs will be submitted to the DSMC through the Velos Clinical Trial Management System. 
Other reportable events: 
Deviations: 
A deviation is a one time, unintentional action or process that departs from the IRB and DSMC approved study 
protocol, involving one incident and identified retrospectively, after the event occurred. If the impact on the 
protocol disrupts the study design, may affect the outcome (endpoints) or compromises the safety and welfare 
of the subjects, the deviation must be reported to the DSMC within 5 business days and the IRB within 10 
business days. 
Prospective Deviations: 
A one time, intentional action (planned prospectively) or process that departs from the IRB and CTSRMC 
approved study protocol, intended for one occurrence. Approval must be obtained from the Sponsor and 
Medical Monitor prior to submitting a prospective deviation request to the DSMC.  IRB and DSMC approval or 
acknowledgment is required prior to implementation. 
 
Events not deemed reportable as outlined above will require a PI assessment regarding study and/or safety 
impact.  This assessment should be documented appropriately. 
11.5 UPENN IRB Notification by Investigator-Sponsor 
The University of Pennsylvania IRB (Penn IRB) requires expedited reporting of those events related to study 
participation that are unforeseen and indicate that participants or others are at increased risk of harm.  The 
Penn IRB will not acknowledge safety reports or bulk adverse event submissions that do not meet the criteria 
outlined below.  The Penn IRB requires researchers to submit reports of the following problems within 10 
working days from the time the investigator becomes aware of the event: 
• 
Any adverse event (regardless of whether the event is serious or non-serious, on-site or off-site) that 
occurs any time during or after the research study, which in the opinion of the principal investigator is: 
Unexpected (An event is “unexpected” when its specificity and severity are not accurately reflected in the 
protocol-related documents, such as the IRB-approved research protocol, any applicable investigator brochure, 
and the current IRB-approved informed consent document and other relevant sources of information, such as 
product labeling and package inserts.) 
AND 
Related to the research procedures (An event is “related to the research procedures” if in the opinion of the 
principal investigator or sponsor, the event was more likely than not to be caused by the research procedures.) 

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Deaths occurring for patients on-study and within 30 days of study drug administration that are considered 
unforeseen and indicates participants or others are at increased risk of harm (i.e. unexpected and 
probably/definitely related), must be reported to the IRB within 24 hours of notification. 
Copies of each report and documentation of IRB notification and receipt will be kept in the Clinical 
Investigator’s study file. 
For clinical drug trials, the following events are also reportable to the Penn IRB: 
• 
Any adverse experience that, even without detailed analysis, represents a serious unexpected adverse 
event that is rare in the absence of drug exposure (such as agranulocytosis, hepatic necrosis, Stevens-
Johnson syndrome). 
• 
Any adverse event that would cause the sponsor to modify the investigators brochure, protocol or 
informed consent form, or would prompt other action by the IRB to assure protection of human subjects. 
• 
Information that indicates a change to the risks or potential benefits of the research, in terms of severity 
or frequency. For example: 
-- An interim analysis indicates that participants have a lower rate of response to treatment than initially 
expected. 
-- Safety monitoring indicates that a particular side effect is more severe, or more frequent than initially 
expected. 
-- A paper is published from another study that shows that an arm of your research study is of no 
therapeutic value. 
• 
Change in FDA safety labeling or withdrawal from marketing of a drug, device, or biologic used in a 
research protocol. 
• 
Breach of confidentiality 
• 
Change to the protocol taken without prior IRB review to eliminate apparent immediate hazard to a 
research participant. 
• 
Incarceration of a participant when the research was not previously approved under Subpart C and the 
investigator believes it is in the best interest of the subject to remain on the study. 
• 
Complaint of a participant when the complaint indicates unexpected risks or the complaint cannot be 
resolved by the research team. 
• 
Protocol violation (meaning an accidental or unintentional deviation from the IRB approved protocol) that 
in the opinion of the investigator placed one or more participants at increased risk, or affects the rights or 
welfare of subjects. 
The IRB will accept other reports when the investigator is unsure whether the event should be reported, and 
the IRB will review such reports to determine whether the event meets the threshold for an unanticipated event 
presenting risk to the participant. 
Institutional Review Board 
3600 Civic Center Blvd., 9th Floor, Philadelphia, PA 19104 
Phone: 215-573-2540 
Fax: 215-573-9438 
 
11.5.1 Reporting Process to IRB at Penn 
 
Principal Investigators are encouraged to submit reports of unanticipated problems posing risks to subjects or 
others using the form: “Unanticipated Problems Posing Risks to Subjects or Others Including Reportable 
Adverse Events” via HS-ERA or a written report of the event (including a description of the event with 

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information regarding its fulfillment of the above criteria, follow-up/resolution and need for revision to consent 
form and/or other study documentation) within 7 working days and the PI or her designee will enter the event 
into HS-ERA and VELOS for Penn and participating sites.  Participating sites should follow local, institutional 
guidelines on Event Reporting. For reportable deaths, the initial submission to the IRB may be made by 
contacting the IRB Director or designee at (T: 215.573.1206).  All deaths will be reported via HS-ERA, as well. 
11.5.2 FDA Notification by Investigator-Sponsor 
 
This study is IND exempt and reporting to the FDA is voluntary using a MedWatch 3500 or via the FDA’s 
website for voluntary reporting. 
11.6 Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study at his site.  This safety 
monitoring will include careful assessment and appropriate reporting of adverse events, as noted above, as 
well as the construction and implementation of a site data and safety monitoring plan.  Medical monitoring by 
an independent clinician Dr. Noelle Frey, Department of Medicine, Division of Hematology-Oncology will 
include a regular assessment of the number and type of serious adverse events on a periodic basis.  Dr. Frey 
will be reached at Noelle.frey@uphs.upenn.edu or via telephone at (215) 662-6901. 
11.7 Study Monitoring Plan 
This study will be monitored by the Principal Investigator and sub-investigators, as appropriate.  Such 
monitoring will include at least weekly meetings of the study team to review accrual, toxicity, SAEs. Dose 
escalations and study finding.  In addition, the PI will ensure that data are completed in a timely manner and he 
or his designee will review the data for accuracy, completeness and integrity.  Further, the PI will view real-time 
toxicity and all laboratory results on an ongoing basis by accessing the Monitoring Report database and the 
Protocol Labs database. 
11.8 Auditing and Inspecting 
The investigator will permit the Cancer Center’s Administrative Director of Compliance and Auditing, or her 
designee to review records, data and facilities at mutually agreeable times. This study will be audited by the 
Department of Compliance and Monitoring (DCOM) on behalf of the Abramson Cancer Center Data Safety and 
Monitoring Committee (DSMC) every six months from the first subject enrolled and every 6 months thereafter 
for the life of the study. The DCOM may modify the frequency of auditing based on acceptable outcomes. 
Scheduling will occur in accordance with the process approved by the National Cancer Institute (NCI) and 
detailed in the NCI approved Institutional Data Safety and Monitoring Plan. 
12.0 STATISTICAL CONSIDERATIONS 
12.1 Phase 1 design 
This is a phase I/exploratory phase II study.  For the phase I portion, the primary objective is determining the 
MTD of HCQ when given in combination with daily nivolumab or nivolumab and ipilimumab in patients with 
advanced melanoma.  Secondary endpoints include response rates, toxicity rates, and pharmacodynamic and 
pharmacokinetic correlative endpoints. Three patients per cohort will be treated for dose escalation in each 
group, and the dose escalation will take a stepwise fashion. If 1 out of 3 patients were defined as having a DLT 
during the phase 1 at a dose level, the cohort will be expanded to 6 patients to obtain the MTD for HCQ within 
this regimen for this patient population. The target DLT rate is 33%. Dose reduction will be pursued if observed 
DLT rate is in excess of 33%. The MTD will be defined as a) the dose producing DLT in < 2 out of 6 patients, 
or b) the dose level below the dose which produced DLT in ≥ 2 out of 3 patients, or in ≥ 3 out of 6 patients 
(Section 6 Tables 3 and 4). 
The table below provides the operating characteristics of the dose escalation plan, as a function of the true 
DLT rate.  For example, if the true DLT rate is 10%, the probability of dose escalation is 91%.  If the true DLT 
rate is 50%, the probability of dose escalation is only 17%. 

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Table 8: Operating Characteristics of the Dose Escalation Plan 
True DLT Rate 
Escalation Probability 
.1 
.91 
.2 
.71 
.3 
.49 
.4 
.31 
.5 
.17 
.6 
.08 
.7 
.03 
.8 
.01 
.9 
.001 
 
12.2 Sample size calculation for phase 2 
Once the MTD has been determined we study the safety and preliminary efficacy in an open label 2 cohort 2 
stage phase 2 trial involving 43 patients with anti-PD1 antibody naive disease (Arm A) and 27 patients with 
anti-PD1 Antibody refractory disease 
 
Cohort 2a (PD1 Ab-refractory): Assuming the null hypothesis of a 5% response rate, a sample size of 27 
patients will provide an 80% power to identify a response rate of 20% with a 0.05 level of significance.  This will 
be a 2 stage design. The first stage will be 13 patients. If 0 patients achieve a PR the study will be terminated. 
If 1/13 or more patients achieve a response the second stage will be activated. If < 3 patients out of 27 patients 
achieve a response no further development of this combination is warranted unless a third compound is added 
 
Cohort 2b (PD1Ab-naïve): Assuming the null hypothesis of a 45% response rate, a sample size of 43 patients 
will provide an 80% power to identify a response rate of 65% with a 0.05 level of significance.  This will be a 2 
stage design. The first stage will be 15 patients: if < 7 patients achieve a PR the study will be terminated; if ≥ 7 
patients achieve a response the second stage will be activated. If < 24 patients out of 43 patients achieve a 
response no further development of this combination is warranted unless a third compound is added.  If 15 
patients are not accrued to the study in 36 months then this arm will be closed due to lack of feasibility. 
 
Cohort 2c (PD-1 Ab-refractory):  Assuming the null hypothesis of a 12% response rate, a sample size of 35 
patients will provide an 80% power to identify a response rate of 30% with a 0.05 level of significance.  This is 
an optimal 2-stage design that provides a 61% chance of stopping early for futility. The first stage will be 11 
patients. If <2 patients achieve a PR the study will be terminated. If 2 or more patients achieve a response the 
second stage of 24 patients will be activated. If <8 patients out of 35 patients achieve a response no further 
development of this combination is warranted.   
 
 
12.3 Analysis of Secondary Endpoints 
For secondary and correlative endpoints, analyses will be primarily descriptive in nature in keeping with the 
study design. All adverse events and clinical responses will be tabulated and summarized. Exact 95% 
confidence intervals will be produced for adverse event and response rates at the MTD.  For all patients 
treated, we will use waterfall plots to present tumor response measured using RECIST 1.1 criteria. We will 
present Kaplan-Meier curves for time-to-event endpoints including PFS for patients at the MTD. We assume, in 
the following plan, that any variables requiring transformation because of skewing or other violations of 
distributional assumptions will be appropriately transformed prior to analysis.  Summary statistics (mean, 
median, standard deviation, range) will be used to describe distributions of various correlative markers at 
baseline and post-treatment timepoints.  Fold change (post-treatment/baseline) of these outcomes will also be 
summarized, and the association with clinical response metrics will be evaluated. The association between 

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continuous outcome measures will be assessed using Pearson or Spearman correlation coefficients. 
Associations between categorical measures (e.g., gender and response) will be assessed using exact tests of 
association implemented in StatXact.  The receiver operating characteristic curve (ROC) and the AUC will be 
computed to evaluate biomarkers that can potentially predict treatment response (e.g. exosomal PDL1).  All 
analyses, except as otherwise noted, will be performed using either StatExact, SAS or STATA. 
 
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APPENDIX A 
 
ECOG PERFORMANCE STATUS* 
Grade ECOG 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or 
sedentary nature, e.g., light house work, office work 
2 
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 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours 
4 
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 ECOG Performance Status is in the public domain therefore available for public use. To duplicate the scale, 
please cite the reference above and credit the Eastern Cooperative Oncology Group, Peter O’Dwyer, MD PhD, 
Group Chair. 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX B 
 
Recommended Assessments by Visit Phase I, II ( See Calendar Section 9 for more details)  
Visit 
Assessments 
Consent to Enroll on study 
History, Physical  
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin, PT, PTT, serum HCG; TSH 
 
Order CT/MRI chest abdomen and pelvis  
 
Tumor biopsy 
Cycle 1 Day 1 
History, Physical  
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin, urine HCG (within 24 hours of treatment); TSH 
 
Research blood draw per section 9 
Cycle 1 Day 12  
Tumor biopsy  
 
Research Blood (See Calendar)  
Cycle 2 Day 1 
History, Physical  
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin,); urine HCG (within 3 days of dosing); TSH 
 
Order CT/MRI chest abdomen and pelvis prior to next cycle  
 
Research Blood (See Calendar)  
Cycle 3 Day 1 
History, Physical  
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin,); urine HCG (within 3 days of dosing); TSH 
 
Research Blood (see Calendar)  
Cycle 4 and beyond  
Repeat Cycle 1-3 as above  
End of Study/Disease Progression  History, Physical  
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin,; TSH 
 
Research Blood (see Calendar)  
 
Tumor Biopsy  
 
 
 
 
 

LIMIT Melanoma Trial  
UPCC 01620 
 
6/13/2023 
Confidential 
64 
 
Recommended Assessments by Visit Phase I (4-drug) (See Calendar Section 9 for details)  
Visit 
Assessments 
 
Consent to Enroll on study 
History, Physical  
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin, PT, PTT, serum HCG; TSH 
 
Order CT/MRI chest abdomen and pelvis 
 
Tumor biopsy 
 
Cycle 1 Day 1 
History, Physical 
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin, urine HCG (within 24 hours of treatment); TSH 
 
Research blood draw per section 9 
 
Cycle 1 Day 12  
Tumor biopsy 
 
Research Blood (See Calendar) 
 
Cycle 2 Day 1 
History, Physical 
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin; urine HCG (within 3 days of dosing); TSH 
 
Research Blood (See Calendar)  
 
Cycle 3 Day 1 
History, Physical  
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin,; urine HCG (within 3 days of dosing); TSH 
 
Research Blood (see Calendar)  
 
Order CT/MRI chest abdomen and pelvis prior to next cycle 
 
Cycle 4  
History, Physical  
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin,); urine HCG (within 3 days of dosing); TSH 
 
Research Blood (See Calendar) 
 
Maintenance Cycle 1  
History, Physical  
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin; urine HCG (within 3 days of dosing); TSH 
 
Research Blood (see Calendar) 
 

LIMIT Melanoma Trial  
UPCC 01620 
 
6/13/2023 
Confidential 
65 
 
Recommended Assessments by Visit Phase I (4-drug) (See Calendar Section 9 for details)  
Visit 
Assessments 
 
Maintenance Cycle 2 
History, Physical 
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin, urine HCG (within 3 days of dosing); TSH 
 
Research Blood (see Calendar) 
 
Order CT/MRI chest abdomen and pelvis prior to next cycle 
 
Maintenance Cycle 3 
History, Physical 
 
EKG 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin,; urine HCG (within 3 days of dosing); TSH 
 
Research Blood (see Calendar) 
 
Maintenance Cycle 4 and beyond 
Repeat Maintenance Cycle 1-3 
 
End of Study/Disease Progression  History, Physical 
 
Troponin, CBC, Sodium, potassium, BUN, serum creatinine, glucose, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, LDH, 
albumin, TSH 
 
Research Blood (see Calendar) 
 
Tumor Biopsy