Efficacy of Low Dose, Subcutaneous Interleukin-2 (IL-2) to Expand Endogenous Regulatory T-Cells in Liver Transplant Recipients
Phase: PHASE2
Conditions: Liver Transplantation
Sponsor: Beth Israel Deaconess Medical Center

NCT Number:  
NCT02739412 
Official Title: 
Efficacy of Low Dose, SubQ Interleukin-2 (IL-2) to Expand Endogenous 
Regulatory T-Cells in Liver Transplant Recipients 
Document Type: 
Protocol with Statistical Analysis Plan 
Document Date: 
09/07/2017 

(CCI) Committee on Clinical Investigations 
Page 1 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
PART B 
STUDY DESCRIPTION 
 
TITLE OF PROTOCOL 
Efficacy of Low Dose, Subcutaneous Interleukin-2 (IL-2) to 
Expand Endogenous Regulatory T-Cells in Liver Transplant 
Recipients 
Principal Investigator 
Michael Curry, MD 
 
B1. 
PURPOSE OF PROTOCOL 
The primary objectives of this pilot study are: 
1. 
To assess the efficacy of very low dose subcutaneous interleukin-2 (IL-2) to promote regulatory 
T cell (Treg) expansion in a cohort of liver transplant recipients with stable graft function, no 
active or history of auto-immune diseases, no evidence of allograft rejection, no current 
evidence of viral hepatitis, and no advanced liver graft fibrosis.  
2. 
To asses the safety and tolerability of very low dose IL-2 in a liver transplant recipient cohort.  
Secondary objectives:   
1. To investigate the percent change of peripheral blood Treg cells and change in phenotype and 
function of Treg cells in response to very low dose IL-2 in a liver transplant recipient cohort. 
2. To identify patients who are IL-2 responsive and thereby potential candidates for withdrawal of 
immunosuppression. This pilot study will not involve withdrawal of immunosuppression (IS), 
however if it is determined that low dose IL-2 is safe, well tolerated, and results in significant 
expansion of Treg cells, then subsequently a follow-up study of IL-2 expansion of Treg cells 
followed by IS withdrawal will be proposed. 
3. Assess short term impact of study treatment on subject’s DSA status, i.e. change in DSA titers or 
development of de novo DSA.   
Exploratory Objective:   
1. Establish baseline phenotypic values for T cell exhaustion in the trial cohort. 
2.   Determine if IL-2 treatment results in development of IL-2 antibodies in the study population 
 
B2. 
SIGNIFICANCE AND BACKGROUND FOR THE STUDY 
Transplantation remains the most successful treatment for end-stage liver disease (ESLD), but the 
need to administer life-long immunosuppression (IS) to prevent rejection limits patient and graft 
survival. Systemic immunosuppression is associated with the development of significant morbidity from 
complicating side effects of hypertension, hyperglycemia and hyperlipidemia contributing to the 
metabolic syndrome and cardiovascular disease, cerebrovascular disease and renal failure. 
Additionally, long-term use of immunosuppression is associated with a remarkably increased risk of skin 
and other malignancies.  Long-term survival following liver transplantation is still suboptimal, and liver 
recipients exhibit higher morbidity and mortality than the general population.  This is mainly due to co-
morbidities arising in large measure from chronic IS treatment.  
Liver transplantation is the only organ transplant setting in which a sizeable proportion of patients 
spontaneously develop “operational tolerance”, a phenomenon defined by the maintenance of stable 
graft function in the absence of destructive immune responses without the need of IS. Withdrawal of 
IS is feasible in selected patients who develop spontaneous operational tolerance, and may 
overcome the problems of exposure to long term IS. Unfortunately, this phenomenon only occurs 
many years after transplantation (i.e. 79% of adult patients >11 years post-transplant, but in 0% of 
adult patients <50 years old and <6 years post-transplant), when irreversible IS-related toxicity has 

(CCI) Committee on Clinical Investigations 
Page 2 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
often already occurred. Therefore, there is an urgent need to develop strategies to intentionally 
induce tolerance in young liver recipients who have been transplanted for a short period of time. 
Immunological tolerance is regarded as an active regulatory process whose outcome depends on the 
balance between cytopathic lymphocytes and graft-protecting, regulatory T cells (Tregs). In animal 
models of transplantation, liver allografts are spontaneously tolerated through regimens that 
dependent on CD4+CD25+Foxp3+ T cells (Tregs). In human liver recipients undergoing IS 
withdrawal years after transplantation, transient intra-hepatic lymphocyte infiltration with preferential 
accumulation of Tregs is also observed. This suggests that a shift in the balance between regulatory 
and effector T cells is critical to achieve tolerance, and that IS withdrawal provides a window of 
opportunity to do so.  
IL-2 is essential for the survival and function of Tregs. In animals, inefficient IL-2 signalling results in 
Treg deficiency and autoimmunity, whereas administration of IL-2 in vivo induces Treg expansion and 
immunoregulation. Deficient IL-2 signaling is a hallmark of human autoimmune diseases such as 
type-1 diabetes. Calcineurin inhibitors (CNI), which constitute the mainstay IS in liver transplantation, 
reduce the overall availability of IL-2. Studies have determined that in liver recipients on CNI, Tregs 
are reduced in number as a consequence of increased apoptosis. Low-dose IL2 reverses this 
phenotype by upregulating the anti-apoptotic BCL-2 gene selectively in Tregs thereby promoting Treg 
expansion.  Tregs, cells that bear high affinity IL-2 receptors, require IL-2 to expand and maintain 
their immunosuppressive phenotype.  Tregs not only expand in blood, but also in peripheral tissues, 
with the largest increase being observed in the liver.  
Recent clinical studies using low dose IL-2 in patients with drug resistant GVHD and certain forms of 
autoimmunity demonstrated that low doses IL-2 (1 million units/m2) is safe and efficacious with a 50% 
response rate in GVHD. Clinical studies performed to date show that even very low doses of IL-2, i.e., 
0.3 million units/m2, expands Tregs whereas doses of 1million units/m2 also expanded the number of 
pro-inflammatory innate immune cells bearing intermediate affinity IL-2Rs.Expansion of pro-
inflammatory cells could counteract, to some degree, the immunoregulatory effects of IL-2 driven 
expansion of Tregs. Therefore, this lower dose (0.3 million IU/m2) would potentially limit this 
unwanted consequence and boost both efficacy and safety.  The affinity of Tregs for IL-2 is much 
higher than innate immune cells except for a tiny, desirable population of highly IL-2 responsive 
immunoregulatory NK cells. Additioanlly, in recent studies of cynomolgus monkey renal transplant 
recipients, with durable drug free graft survival, rejection was incited by daily treatment with 1 million 
IU/ m2. IL-2 induced an unwanted expansion of memory T cells. Taken together, the results in clinical 
and monkey studies indicate that in treatment with 1 milion IU/ m2 IL-2, levels of IL-2 hover in a range 
where Tregs expand, but this is accompanied by unwanted activation of innate immune cells and/or 
T-memory, such that the stimulatory effect on Tregs is offset, sometimes moderately, sometimes 
markedly, by activation and expansion of inflammatory innate and memory T cells.  
In consideration of these most recent findings, we amend the protocol (version date 9.7.2017), to 
reduce the dose of IL-2 (from 1.0 million to 0.3 million units/m2) to expand Tregs, at a dose that does 
not expand pro-inflammatory immune cells to further improve the safety profile while still promoting 
tolerance. 
This provides additional rationale for administering a very low-dose IL-2 to liver transplant recipients 
to expand the pool of endogenous Tregs and shift the balance between effector and regulatory 
lymphocytes at the time IS drugs are discontinued.  
To maximize the benefit derived from IS withdrawal, there is a need to find strategies to intentionally 
induce tolerance in young recipients in whom accumulated IS toxicity has not yet occurred. Tregs can 
suppress cytopathic immune responses and inhibit transplant rejection. In contrast to innate immune 
cells and effector T cells, resting Tregs constitutively express the high affinity IL-2 receptor, making 
Tregs exquisitely sensitive to very low-doses of IL-2. This protocol proposes to employ low-dose IL-2 
to promote the selective expansion of endogenous Tregs and shift the balance between effector T 
cells and Tregs. Studies have revealed that successful IS discontinuation is associated with a 

(CCI) Committee on Clinical Investigations 
Page 3 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
transient intra-graft immune regulatory response with preferential accumulation of Treg cells. This 
suggests that short-term enhancement of Treg numbers and/or function at the time of IS withdrawal 
may facilitate the acquisition of tolerance in patients who are not predisposed to spontaneously 
develop it. IL-2 is a cytokine that is essential for the optimal development, survival and function of 
Tregs. Several clinical studies have shown that low-dose IL-2 preferentially expands Tregs and is 
safe and efficacious in patients with autoimmunity or graft verses host disease (GVHD). In these 
studies, Treg frequency increased up to 2 to 8-fold without significant changes in the number of 
effector T cells. Our objective is to investigate if administration of a short-course of low-dose IL-2 to 
liver transplant recipients facilitates an increase in peripheral blood Treg cells. We propose to conduct 
this pilot study to assess safety and efficacy of IL-2 in expanding peripheral blood Treg cells and 
assess the effect of IL-2 on T effector cells and cell of the innate immune response in these transplant 
recipients.  
The primary objectives of this pilot study are: 
1. 
To assess the efficacy of very low dose subcutaneous interleukin-2 (IL-2) to promote regulatory 
T cell (Treg) expansion in a cohort of liver transplant recipients unlikely to be safely withdrawn 
from from IS in the absence of other treatment with stable graft function, no active or history of 
auto-immune diseases, no evidence of allograft rejection, no current evidence of viral hepatitis, 
and no advanced liver graft fibrosis.  
2. 
To asses the safety and tolerability of very low dose IL-2 in a liver transplant recipient cohort;  
3. 
Assess short term impact of study treatment on subject’s DSA status, i.e. change in DSA titers or 
development of de novo DSA. 
Secondary objectives:   
1. 
To investigate the percent change of peripheral blood Treg cells and change in phenotype and 
function of Treg cells in response to very low dose IL-2 in a liver transplant recipient cohort; 
2. 
To identify patients who are IL-2 responsive and thereby potential candidates for withdrawal of 
immunosuppression. This pilot study will not involve withdrawal of immunosuppression (IS), 
however if it is determined that very low dose IL-2 is safe, well tolerated, and results in 
significant expansion of Treg cells, then subsequently a follow-up study of IL-2 expansion of 
Treg cells followed by IS withdrawal will be proposed. 
Exploratory objective: 
1. Establish baseline phenotypic values for T cell exhaustion in the study cohort. 
2. Determine if IL-2 treatment results in development of IL-2 antibodies in the study population   
References: 
Benítez C. Multicenter clinical trial of immunosuppression withdrawal in liver transplantion. 
Hepatology 2013;58(5):1824-35 
Bohne F. Intra-graft gene expression predicts the development of tolerance in liver transplantation. J 
Clin Invest 2012;122(1):368-82 
Hartemann A. Low-dose interleukin-2 in patients with type-1 diabetes. Lancet Diabetes Endocrinol 
2013 Dec;1(4):295-305. 
Koreth J. Interleukin-2 and Regulatory T Cells in GVHD. N Engl J Med. 2011;365(22):2055-66 
Matsuoka K. Interleukin-2 restores regulatory T cell homeostasis in chronic GVHD. Sci Transl Med 
2013;5(179):179ra43 
Yamada Y, Nadazdin O, Boskovic S, Lee S, Zorn E, Smith RN, Colvin RB, Madsen JC, Cosimi AB, 
Kawai T, Benichou G. Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed 
Hematopoietic Chimerism in Monkeys. Am J Transplant. 2015 Dec 1;15(12):3055–3066.  

(CCI) Committee on Clinical Investigations 
Page 4 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
 
B3. 
DESCRIPTION OF RESEARCH PROTOCOL 
A. 
Study Design – Overview, Methods, Procedures 
The study is a single arm, prospective, open label efficacy trial of a 4 week course of low dose 
subcutaneous IL-2 (1MIU/mL/m2/day) to expand peripheral blood regulatory T cells (Tregs) in a liver 
transplant population..   
This pilot trial will treat up to 10 liver transplant recipients who are ≤65 years of age, a minimum of 2 
years but no more than 4 years post primary liver transplantation for non-autoimmune conditions, have 
had no evidence of transplant rejection, and have no current evidence of liver allograft dysfunction or 
viral hepatitis. 
Study objectives include (1) determine the efficacy of the administration of low dose IL-2 to promote 
Treg cell expansion; (2) asses the safety and efficacy of low dose IL-2 in a liver transplant recipient 
cohort; (3) investigate the percent change of peripheral blood Treg cells and change in phenotype and 
function of Treg cells in response to low dose IL-2 in a liver transplant recipient cohort. 
 
 
Study Scheme:   
 
 
  
 
Weeks: 
 
0 
1 
2 
3 
4 
8 
12 
 
       36 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study Procedure: 
Screening (S0):  
After obtaining informed consent the following screening procedures will be completed: 
 Full history and medical record review 
 Physical examination including height and weight  
 Vital signs (blood pressure, heart rate and temperature) 
 Blood (approximately 20 milliliters) will be drawn for  
 liver function panels (AST, ALT, ALP, Bilirubin, Albumin) and kidney function panels (Na, 
K, Cl, CO2, Creatinine, Glucose, Ca) if not previously obtained within 1 month of 
screening 
 viral hepatitis and autoimmune panels (HBV sAg, HCV Ab, ANA, ASMA IgG) if not 
previously obtained within 6 months of screening 
 serum pregnancy test for women of child bearing potential (WOCBP) 
 Fibroscan if liver biopsy/fibroscan results not obtained within 1 year of screening 
 ECG if not obtained within 3 months of screening 
Subjects with liver panel results <2xULN, serum creatinine <1.5 ULN; no evidence of viral hepatitis or 
IL-2 injections each day x 4 weeks 
SOT  
EOT 
EOS 
Efficacy & Safety Measures 
Follow-up Measures 
SOT: Start of Treatment 
EOT: End of Treatment 
EOS: End of Study  

(CCI) Committee on Clinical Investigations 
Page 5 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
autoimmunity, no evidence of pregnancy, absence of advanced liver fibrosis, no history of seizure 
disorder and no evidence of cardiac disease will proceed with the study (see Inclusion/Exclusion  
criteria in section below). 
Baseline/Treatment Visit Day 1 (W0): 
Subjects will return to clinic to complete baseline measures and begin study treatment:  
 Review of any new health problems, current medication and vital sign measures; 
 Blood draw (approximately 35 milliliters) for  
 CBC with differential,  
 liver and kidney function panels, 
 immune cell counts , 
 T cell exhaustion phenotyping, 
 serum IL-2 levels, 
 CNI trough levels, 
 presence of donor specific antibodies (DSAs)  
 presence of anti-IL-2 antibodies 
 
Treatment: Subjects will start very low dose IL-2 subcutaneous injections at a once per day dose of  
0.3 million IU/mL per square meter body surface area (BSA) for 4 weeks. Study staff will instruct 
subjects and/or identified care providers (e.g. friend or spouse) on subcutaneous injection technique.  
The first study dose will be administered in the BIDMC Harvard-Thorndike Clinical Research Center.  
Subjects will remain in house for 2 hours of observation to monitor drug tolerability and adverse events. 
Subjects will be given a paper calendar to record their self-administration of the study drug.  Subjects 
will record the time of injections each day and bring the study calendar and all unused drug to all study 
visits. (see Calendar attached) 
Treatment (D3): 
Subjects will return to clinic on Day 3 for evaluation of adherence to treatment and staff observation of 
proper injection technique (for Dose 3) and the following measures will be obtained: 
 
Review of any new health problems, current medications  
 
Vital signs  
 
Blood draw (approximately 10 milliliters) for liver and kidney function panels 
 
Treatment (W1, W2, W3, W4) 
Subjects will return to clinic weekly until Day 28 (Week 4).  Subjects will receive a refill of IL-2 (not at 
Week 4 visit) and the following measures will be obtained: 
 Review of any new health problems, current medications, vital signs and weight,  
 ECG, 
 Blood draw (approximately 35 milliliters) will be drawn for  
 CBC with differential, 
 liver and kidney function panels, 

(CCI) Committee on Clinical Investigations 
Page 6 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
 serum IL-2 levels, 
 immune cell counts (Weeks 2 and 4)   
 Week 4 only: CNI trough levels and serum pregnancy test (WOCBP) 
 DSAs (Week 4) 
 anti-IL-2 antibodies  (Week 4) 
 
Subjects will discontinue IL-2 treatment after Day 28 (Week 4). 
Subjects will have blood drawn for standard of care labs at Week 4, i.e. CBC, liver and kidney panels, 
and CNI trough levels.  We will obtain standard of care results from subjects’ medical records and draw 
extra blood (approximately  25 ml ) for research testing for DSA and anti-IL-2 antibodies (week 4), 
serum IL-2 levels and PBMCs flow cytometry and a serum pregnancy will be conducted for WOCBP.  
Follow-up (W8, W12, W36) 
Subjects will return to clinic for post-treatment follow-up at Weeks 8, 12 and 36.  The following 
measures will be obtained: 
 Review of any new health problems, current medications, vital signs and weight,  
 Full physical examination will be completed at end of study (Week 36); 
 Review standard of care lab results for 
 CBC with differential, 
 liver and kidney function panels, 
 CNI trough levels, 
 Blood draw (approximately 20 milliliters) for study labs 
 Immune cell counts  (Weeks 8 and 12) 
 serum IL-2 levels (Weeks 8 and 12) 
 serum pregnancy test for WOCBP (Weeks 8 and 12). 
 anti-IL-2 antibodies (Week 36)  
Dosage Reduction and Treatment Withdrawal  
The dose of IL-2 may be reduced by half to   0.15 million IU /m2 daily if a subject experiences untoward 
side effects.  If symptoms improve, the dose may be re-escalated to target.  If symptoms persist, the 
dose frequency may be reduced to every other day administration or the Investigator may discontinue 
study treatment.  
Study treatment will be discontinued in the presence of any of the following circumstances:  
 Subject withdraws consent  
 Life threatening anaphylactic reaction to IL-2 
 Life threatening infection on IL-2 
 The Investigator may discontinued study treatment in the presence of other clinically significant 
findings including abnormal ECG, blood panel results >2x ULN and/or other signs of significant 
toxicities.  
Subjects who are withdrawn from treatment will complete remaining study visits and continue to receive 
follow-up care from his/her BIDMC transplant Hepatologist. 

(CCI) Committee on Clinical Investigations 
Page 7 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
Standard of Care Maintenance Immunosuppression: Eligible patients must be receiving stable 
dosing of immunosuppressant therapy for a minimum of 1 month prior to study enrollment.  
Maintenance immunosuppression is customize to the patient but typically, stable liver transplant 
patients at 2-4 year post transplant who are otherwise eligible to enroll into this trial would be taking 
tacrolimus (target 12 hour trough goal of 4-6ng/mL) with or without mycophenolic acid (as 
mycophenolate mofetil or mycophenolate sodium) at a dose between 500-1000mg twice a day.  
 
Safety Monitoring  
The PI is ultimately responsible for protecting the rights, safety and welfare of subjects enrolled into the 
trial. Study visits will be conducted weekly during the treatment phase of the trial; post treatment follow-
up visits will be conducted at week 8 and 12.  The PI will review all safety parameters including clinical 
safety laboratory assessments within 48 hours of completion of the measure.  Clinical laboratory values 
that fall outside of the pre-specified safety ranges will be brought to the PIs attention the day the reports 
are final to assess clinical significance.   
Patients of the Transplant Institute have direct access to Transplant Nurse Coordinator consultation 
24/7/365.  Transplant Coordinators will alert the PI or designee for any study subject who calls with 
questions or concerns.  
Patients will be given a Study Information card and instructed to present it to any health care provider 
they see during the time that are participating in the trial.  The Study ID card will include a brief 
description of the trial, name of the study drug, and PI contact information. See safety card attached.  
 
10.2  Monitoring for AEs  
Subjects will be monitored for the occurrence of adverse events at all study visits, including follow up.   
 
Reporting procedures will comply with BIDMC IRB policies; US Federal Regulations (21CFR 312), and 
ICH Guideline E-6: Guidelines for Good Clinical Practice. 
 
Adverse events will be coded and graded according to the NIH/NCI Common Terminology Criteria for 
Adverse Events version 4.03 (June 2010).  All adverse events will be classified on seriousness (mild, 
moderate, serious), expectedness (expected, non-expected), and relatedness to trial (not related, 
possibly related, related).   
 
Reporting Serious Adverse Events:  All serious adverse events (SAEs) will be reported to the BIDMC 
IRB, regardless of the determined relationship to the study drug, within the timeframes specified below:  
 
Unexpected Fatal or Life-Threatening SAEs will be reported within one (1) business day of PI 
awareness. A formal written report (signed by the PI) will be submitted within seven (7) calendar 
days of PI awareness.  
 
All other unexpected SAEs will be reported as a formal written report (signed by the PI) within 
seven (7) calendar days of PI awareness. 
 
Expected SAEs: For all expected SAEs, we will submit a formal, written report (signed by the 
PI) with fourteen (14) calendar days of PI awareness.   
 
Non-serious AEs will be evaluated by the PI to determine the relationship of the event to the study 
drug, then reported to the CCI in the timeframe and manner specified below:  
 
Related: Non-Serious AEs determined by the PI to be possibly, probably, or definitely related to 
the Study (or relationship unknown):  

(CCI) Committee on Clinical Investigations 
Page 8 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
 Unexpected/related: non-serious AEs that are unexpected and determined to be related to 
study drug, will be reported in a formal written report (signed by the PI) within fourteen 
(14) calendar days of PI awareness.  
 Expected/related:  non-serious AEs that are expected and determined to be related but 
which occur with greater frequency than expected, will be summarized for submission on 
the continuing review progress report at the study’s continuing review.  
 
Unrelated: Non-serious AEs determined by the PI to be unrelated to the study: 
 Unexpected /unrelated: non-serious AEs that are unexpected and determined to be 
unrelated which occur with greater frequency than expected, will be summarized for 
submission on the continuing review progress report at the study’s continuing review 
IND Safety Reporting  
In compliance with federal regulation (21 CFR 312), safety reports will be submitted to  the FDA (via 
submission of an IND Safety Report) when an event meets all of the following criteria: 
 
Suspected adverse reaction (reasonable possibility of relationship with study drug)  
 
Serious 
 
Unexpected  
Fatal or life threatening events will be reported as soon as possible but no later than 7 calendar days 
from time the Sponsor becomes aware.   
Serious and unexpected adverse events Adverse Events will be reported as soon as possible but no 
later than 15 days from the time the Sponsor becomes aware.   
 
Reporting to Prometheus  
 
A copy of any SAE report submitted to the BIDMC IRB, Sponsor and FDA will be sent to Prometheus 
Laboratories.  If interleukin-2 is a suspect or co-suspect drug reported on the FDA Form 3500A 
MedWatch Report, a copy of the FDA Form (3500A MedWatch Report) will also be sent to 
Prometheus Laboratories as a courtesy.   Reports to Prometheus will be sent to Drug Safety and 
Pharmacovigilance at Prometheus Laboratories Inc. All contact information will be included in the 
Report.  
 
Drug Safety Email: 
drugsafety@prometheuslabs.com 
Drug Safety fax: 
(858) 754-3046  
Avoiding bias in laboratory testing:  
Blood specimens drawn and processed for immunological testing will be de-identified, logged and 
labeled prior to transfer to the central laboratory.  Specimen labeling system will be designed such 
that the lab staff will not be aware of the visit sequence of the specimen in relation to pre vs post 
treatment status.  
Independent Medical Monitor (IMM)  
The PI has named an Independent Medical Monitor who is familiar with the agent and with the patient 
population, Simon Robson, MD, PhD, Professor of Medicine, Harvard Medical School. Dr. Robson is 
a world leader in the study of vascular and immune cell ectonucleotidases in pathophysiology and 
transplantation. He is also a prominent investigator in liver and gastrointestinal diseases.  
The IMM will review all SAE reports sent to the BIDMC IRB, Sponsor and FDA in real time.  The IMM 
will also review aggregate data as they are available.   
 

(CCI) Committee on Clinical Investigations 
Page 9 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
Routine Monitoring: To further ensure the study is conducted and documented in accordance with 
the investigational plan, applicable regulations and good clinical practices, routine monitoring will be 
conducted by the Transplant Institute’s Clinical Research Associate or other qualified designee in 
fulfillment of the Sponsor’s obligation for monitoring. Monitoring activities will include:  
 
Review of essential documents in the regulatory binder for completeness and currency;  
 
Inspection of every (100%) informed consent form to assure that correct versions are used; 
that the names of subject and consenter are legible; that forms are properly signed and dated 
by subject and consenting physician investigator; and documentation is available to document 
consent procedures.  
 
Verification that every enrolled subject meets eligibility criteria;  
 
Review of a minimum of 10% of case report forms entered to source verify accuracy and 
completeness with specific attention to … 
 Dose modifications 
 Adverse events, concomitant medications and intercurrent illnesses 
 Missed study visits, procedures, labs, etc 
 Drop out and withdrawals  
 
Review of all regulatory reporting  
 
Reporting findings to Sponsor/Investigator.  
 

(CCI) Committee on Clinical Investigations 
Page 10 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
 
 
Table 1: Study Visits Schedule and Procedures  
 
Screening and 
Baseline 
Treatment to Week 4 
Follow-up to Week 36 
Visit 
Screening 
Baseline 
Day 1 
Day 3 
W1 
W2 
W3 
W4 
W8 
W12 
W36 
Study Procedures: 
Visit # 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
Informed Consent 
√ 
 
 
 
 
 
 
 
 
 
Full Physical Exam & Medical History 
√ 
 
 
 
 
 
 
 
 
√ 
Limited Physical Exam & Medical History 
 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
 
Vital Signs 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Electrocardiogram (ECG) 
√ 
 
 
√ 
√ 
√ 
√ 
 
 
 
Fibroscan  
√1 
 
 
 
 
 
 
 
 
 
Study Blood Tests:  
 
 
 
 
 
 
 
 
 
 
Pregnancy test (for women of child bearing potential) 
√ 
 
 
 
 
 
√ 
√ 
√ 
 
CBC with Differential  
 
√ 
√ 
√ 
√ 
√ 
√4 
√4 
√4 
√4 
Liver panel (AST, ALT, AP, Total Bilirubin, Albumin) 
√2 
√ 
√ 
√ 
√ 
√ 
√4 
√4 
√4 
√4 
Kidney panel (Na, K, Cl, CO2, Creatinine, Glucose, Ca) 
√2 
√ 
√ 
√ 
√ 
√ 
√4 
√4 
√4 
√4 
Viral hepatitis and autoimmunity panels (HBVsAg, HCVAb, ANA, ASMA IgG) 
√3 
 
 
 
 
 
 
 
 
 
Immunosuppression drug level  
 
√ 
 
 
 
 
√4 
√4 
√4 
√4 
Immune cell counts  
 
√ 
 
 
√ 
 
√ 
√ 
√ 
 
IL-2 Blood level 
 
√ 
 
√ 
√ 
√ 
√ 
√ 
√ 
 
T-cell exhaustion markers (only at baseline) 
 
√ 
 
 
 
 
 
 
 
 
DSA (pre and post IL-2 treatment) 
 
√ 
 
 
 
 
√ 
 
 
 
Samples anti IL2 antibody testing 
 
√ 
 
 
 
 
√ 
 
 
√ 
  1 Only if a fibroscan or a liver biopsy was not completed for standard of case within the last 12 months  
  2 Only if liver panel or kidney panel was not collected for standard of care within the last month 
  3 Only if viral and autoimmunity tests not completed for standard of care within past 6 months   
  4 Obtained as part of standard of care  
 

(CCI) Committee on Clinical Investigations 
Page 11 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
 
B. 
Statistical Considerations 
Sample Size Justification:  This is a pilot study to determine the efficacy and safety of very low dose 
IL-2 administration in a select group of liver allograft recipients.  Investigators will consent as many as 
12 subjects into the screening phase in order to treat not more than 10 patients from the BIDMC 
Transplant Institute’s Liver Transplant Program. 
The primary endpoint for the trial is the percent change in absolute numbers of regulatory T cells 
(Tregs) from pre-treatment baseline to end of treatment, i.e. the week 4 treatment visit.  
For IL-2 to be clinically viable, we would require evidence that 60-80% of participants experience a 
biological response defined as a 2 fold increase in absolute number of Treg cells from baseline.  Less 
than 50% of responding participants would be an unacceptable low response. The success of IL-2 will 
be defined based on a one sided 95% confidence interval (CI). The CI for a 0.80 response rate with 10 
subjects would be 0.59-1.00 enabling exclusion of a response rate of 0.50. 
Data Analysis:  Differences in absolute numbers of circulating endogenous Treg cells from baseline 
to end of treatment will be compared using standard descriptive statistics and paired t-tests.  
 
C. 
Subject Selection 
Study subjects will be identified from the BIDMC Transplant Institute Liver Transplant Program by 
hepatologist Michael Curry, MD (Medical Director of the Liver Transplant Program) and surgeon Khalid 
Khwaja, MD (Surgical Director of the Liver Transplant Program).   
Inclusion criteria: 
1. Adult liver transplant recipients 2-4 years post trapslantation 
2. Male or female, age ≤ 65 years old 
3. Stable dosage of immunosuppressant therapy for 1 month prior to study. 
Exclusion crtieria: 
1. Recipient of multiple transplants (including solid organ, stem-cell, and bone marrow) 
2. Serum liver panel (ALT, AST, Alkaline Phosphatase and Total Bilirubin) > 2 x ULN, 
3. Serum creatinine > 1.5 x ULN, 
4. eGFR of < 40 ml/min, 
5. Detectable hepatitis viral load, 
6. Abnormal ECG with clinically significant findings per study physician’s judgement, 
7. Active infection, 
8. Presence or history of autoimmunity disorders, 
9. Evidence of allograft rejection, 
10. Liver biopsy or fibroscan evidence of advanced stage liver fibrosis (> Stage 2 Fibrosis),  
11. Presence or history of cardiac or pulmonary disease,  
12. Pregnant or nursing (lactating) women, 
13. Presence or history of seizure disorder, 
14. Health condition precludes participation in trial at study physician’s judgment, 
15. Inability to give consent. 
 
Male and female participants of all racial distributions are eligible for this study. Participation is limited to 
subjects who are less than 4 years post-transplant and less than or equal to 65 years old as individuals 
who are further out from transplant and older in age are likely to have a higher chance of spontaneous 
operational tolerance and less likely to need pharmacologic intervention to achieve tolerance. 

(CCI) Committee on Clinical Investigations 
Page 12 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
  
B4. 
POSSIBLE BENEFITS 
It is not possible to predict whether subjects will benefit directly from participation in this study.  
Regardless, participation may help others in the future as a result of knowledge gained from the 
research.  
Demonstration that very low dose, subcutaneous IL-2 can induce endogenous Treg cell expansion will 
allow further study of IL-2 mediated operational tolerance in liver allograft recipients.   Future studies will 
consider reducing or withdrawing systemic IS in a cohort of patients who respond to IL-2 expansion of 
Tregs.  
This preliminary study may identify patients who are IL-2 responsive and possible candidates for future 
studies of IS reduction. Successful induction of tolerance and withdrawal of IS would decrease the 
morbidity associated with lifelong use of IS agents for control of organ rejection. 
 
B5. 
POSSIBLE RISKS AND ANALYSIS OF RISK/BENEFIT RATIO 
Risk of Interluekin-2 (Proleukin, IL-2): 
IL-2 is approved as a treatment for metastatic renal cell carcinoma and metastatic malignant 
melanoma. It is most commonly administered to cancer patients as a high-dose intravenous 
treatment regimen. For these cancer indications, dosing guidelines prescribe a three times per day 
intravenous infusion of 600,000 International Units (IU) per kilograms of body weight (e.g. dose for a 
person weighing 82kg is 49.2 million IU x 3 infusions/day = 147.6 million IU per day, compared to a 
daily dose for the same person for the proposed protocol would = 1.95 million IU/day)   Significant 
side effects are expected with the high dose IV regimen for cancer treatment though they are 
reversible once treatment is stopped. Type and severity of side effects is dose dependent.  
The drug manufacturer Novartis has conducted 7 Phase I/II clinical trials in 235 patients with a variety 
of doses and schedules using subcutaneous IL-2.  Dosages have ranged from 2 million IU per day to 
9 million IU per day.  The study populations included metastatic renal cell carcinoma, advanced 
malignancy, and chronic lymphocytic lymphoma.  In these trials, the following adverse events were 
recorded: 
 
Pyrexia (fever) – 77% 
 
Rigors (shivering) – 68% 
 
Asthenia (tiredness) – 49% 
 
Anorexia (loss of appetite) – 54% 
 
Nausea – 50% 
 
Vomiting – 49% 
 
Diarrhea – 40% 
 
Headache – 35% 
 
Dry skin – 25% 
 
Rash – 24% 
 
Injection site inflammation – 24% 
 
Dyspnea (shortness of breath) – 24% 
 
Injection site reaction – 24% 
 
Cough – 23% 
 
Myalgia (muscle pain) – 23% 
 
Insomnia – 21% 
 
Hypokinesia (muscle rigidity) – 20 % 
 
Dizziness – 18% 
 
Abdominal pain – 16% 
 
Injection site pain – 14% 

(CCI) Committee on Clinical Investigations 
Page 13 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
 
Depression – 11% 
 
Pruritus (itchiness) – 10% 
 
Oliguria (decrease in urine production) – 9% 
 
Confusion – 8% 
 
Hypotension (low blood pressure) – 7% 
 
Anxiety – 6% 
 
Increased serum creatinine – 4% 
 
Tachycardia (increased heart rate) – 3% 
 
Gastrointestinal bleeding – 2% 
 
Arrhythmia (unusual heart beat)– 1% 
 
Myocardial infarction (heart attack) – <1% 
 
Kidney failure – <1% 
The most common serious adverse event associated with high dosage, IV administered IL-2 is 
capillary leak syndrome where capillary permeability increases and serum proteins and fluids move to 
the extravascular space.  In subcutaneous administration of IL-2, capillary leak syndrome is much 
less frequent and less severe.  In the Novartis clinical trials of subcutaneous IL-2, no cases of 
pulmonary edema were recorded.  Peripheral edema occurred in 14% of patients and unspecified 
edema occurred in 11% of patients. 
IL-2 has not been approved for use for the indication being tested in this study. In this study we will 
test the efficacy and safety of low dose IL-2, i.e.,   0.3 million IU per square meter body surface area 
(BSA) given once a day in subcutaneous injections, to liver transplant patients over a 4 week period 
of time to measure the effect on the number of peripheral Treg cells. 
Low dose IL-2 has been tested in other clinical trials and similar adverse effects are expected in this 
trial.  A dose escalation trial of IL-2 conducted at Dana Farber Cancer Institute tested safety and 
efficacy of low dose IL-2 at three dose levels, i.e., 0.3 million IU, 1 million IU, and 3.0 million IU (per 
body square meter per day) for treatment of chronic Graft vs Host Disease that was refractory to 
standard care (Koreth et al, 2011).  IL-2 dose (0.3MIU/m2) for 3-4 weeks promoted the expansion of 
Tregs without expanding innate immune cells, however, study protocol required dose escalation to 
maximum tolerated dose and thus the 0.3 MIU/m2 dosing was not formally tested for clinical efficacy. 
29 subjects were enrolled; one patient withdrew early. The following treatment related adverse events 
were reported in the 28 patients who could be evaluated:   
 
Induration (skin hardening) – 11% 
 
Thrombotic microangiopathy (kidney failure) – 7% 
 
Flu-like symptoms – 4% 
 
Increase in serum creatinine – 4% 
 
Thrombocytopenia (platelet deficiency) – 4% 
Another clinical trial in Paris, France studied low-dose subcutaneous IL-2 at a dose of 1.5 million IU 
per day and a dose of 3 million IU per day in 10 Hepatitis C vasculitis subjects (Saadoun et al, 2011).  
No adverse events were reported at a dosage of 1.5 million IU per day and the following adverse 
events were reported at a dosage of 3 million IU per day: 
 
Injection site reaction – 50% 
 
Asthenia (tiredness) – 40% 
 
Flu like symptoms – 40% 
 
Myalgia (muscle pain) – 10% 
 
Hypertension (high blood pressure) – 10% 
 
Infection – 10% 
Based on signs and symptoms observed in the first subject enrolled into this trial (2016P-00086), 
three adverse events are added to the list of expected adverse events, i.e. eosinophilia, mouth ulcer 

(CCI) Committee on Clinical Investigations 
Page 14 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
and pharyngitis (sore throat).  
Contraindications: Patients will be counselled not to undergo routine radiological studies that require 
the use of iodine containing contrast agents during the duration of the study – including follow up (8 
weeks after final dose).  There is a possible increased risk of adverse reaction to the contrast agents 
which include fever, chills, nausea, vomiting, pruritus, rash, diarrhea, hypotension, edema, and 
oliguria.  Most reactions occur within 4 weeks of taking IL-2. 
Patients will also be informed of the listed contraindication of use of high dose IL-2 in organ transplant 
patients when used for FDA approved treatments involving the higher dose IV administrations for 
metastatic melanoma and renal cell carcinoma.  NB: While there is a theoretical risk of transplanted 
organ rejection with high dose IL-2 treatments, there have been no documented cases of organ 
rejection reported with use of IL-2.  
Although the drug manufacturer recommends against use of high dose IL-2 for patients with organ 
transplantation, participants in this study will take low dose IL-2 as this study is testing if very low 
dose IL-2, taken by healthy liver transplant patients will increase the number of T-reg cells in the 
blood.  If this study discovers that low dose IL-2 does increase T-reg cells, Investigators will plan a 
follow-up study to investigate if healthy liver transplant patients develop ‘tolerance’ for their 
transplanted liver.  Patients who develop ‘tolerance’ may be candidates to decrease or discontinue 
anti-rejection medications in a follow up study.  
Use of any live vaccination is contraindicated in patients taking IL-2.  At every visit staff will review all 
concomitant medications with patients and patients will be instructed to report to study staff all 
concomitant drugs, including prescription drugs, vaccinations, over the counter medications, vitamins 
and supplements. Further, patients will be instructed to tell all other practitioners that they are 
participating in a clinical trial and show those practitioners the Study Information Card.  Patients will 
also be instructed to check with the Study Doctor prior to taking any new prescribed medication, 
vaccination, OTC medication, vitamins or supplements.  
PREGNANCY AND FERTILITY:  
Because the effects of interleukin-2 on the developing fetus are not known, subjects may not 
participate in this study if they are pregnant or breastfeeding.  Women capable of becoming pregnant 
will be required to take a pregnancy test to verify that they are not pregnant before receiving their first 
dose of IL-2. 
Furthermore, women capable of becoming pregnant must agree to use adequate birth control for the 
duration of the study.  For the purpose of this study, use of adequate birth control includes one of the 
following: oral hormonal contraceptives, implanted hormonal contraceptives, diaphragm with 
spermicide, Intrauterine device, condoms used with another form of contraception as listed above, 
and abstinence. 
Men capable of fathering children must use adequate contraception while participating in this study.  
For the purposes of this study, adequate birth control means use of a condom and/or partner must 
use an approved method of birth control as listed above. 
The effect of IL-2 treatment on fertility.  It is also unknown what effect IL-2 may have on future 
pregnancies. 
Study subjects who become pregnant while participating in this study must inform study investigators 
immediately.  Subjects will be given a pregnancy test.  If the results demonstrate that the subject is 
pregnant, the subject must withdraw from the study, and the study investigators will ask to monitor the 
pregnancy.   
Risk of Blood Draw: 
Blood samples are collected as a part of this research study.  The risks involved in drawing blood from 
a vein may include, but are not limited to:  

(CCI) Committee on Clinical Investigations 
Page 15 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
 
Momentary discomfort where we draw the blood from, 
 
Bruising and swelling around the site where we draw the blood from,  
 
A feeling of lightheadedness when the blood is drawn, and  
 
Rarely an infection where we draw the blood from.  
Risk of ECGs: 
Subjects might experience temporary redness or a rash on the skin where the adhesive pads are 
placed.  
Confidentiality:  
There is the potential for loss of confidentiality by participating in this study.  Every effort will be made to 
protect the confidentiality of subject’s identifiable information.  
 
B6. 
RECRUITMENT AND CONSENT PROCEDURES 
Recruitment: Study participants will be identified from among the ranks of patients who receive post-
transplant care in the BIDMC Liver Transplant Program.  Once the protocol is approved, Drs. Curry 
and Khwaja will identify patients within the practice who meet preliminary eligibility criteria. Patients 
who meet preliminary criteria will be contacted by an Investigator to come to the TI to learn more 
about the trial, complete consent procedures and requisite screening measures to confirm eligibility. 
Consent: Consent procedures will be completed for patients in a private setting, e.g. Transplant 
Center examination room, consultation room or Investigator’s Office. A study investigator will review 
all study procedures, and risks and benefits will be discussed.   
The patient will be given ample time to ask questions and all his/her questions will be answered. If the 
patient prefers to take the consent home to think about it and discuss participation with others, 
consent procedures will be completed at a subsequent visit.   
Patients willing to consent to participation will be required to sign and date the BIDMC CCI approved 
consent; the Investigator completing consent procedures will also sign and date the consent 
document. The original consent will be maintained in the subject’s research record, a copy will be 
placed in the subject’s medical record, and a copy of the signed consent will be given to the subject.  
Subject Protection: We do not believe participants are vulnerable to coercion or undue influence. 
 
B7. 
STUDY LOCATION 
Privacy: Consent will customarily be completed in an LMOB out-patient clinic exam room. In some 
instances, consent procedures may be completed in a hospital in-patient setting such as a hospital in-
patient bedroom. Consent procedures will always be completed in a private setting beyond the 
earshot of uninvolved staff or other patients.  Study measurements, special tests and blood and urine 
samples will be collected by BIDMC staff at BIDMC facilities where confidentiality and patient privacy 
is upheld through hospital policy. Only the minimal amount of information needed to accomplish the 
research purposes will be collected. 
Physical Setting: Interactions with study participants will take place in a medical setting at BIDMC 
such as the CRC for first dosage observation and the Transplant Clinic in LMOB7 when patients 
come for standard of care visits. There may be occasions where study visits are completed in other 
clinical or hospital locations including a hospital in patient bedroom.   
 
B8. 
DATA SECURITY 
Study staff is trained in human subject protections regulation including hospital mandated CITI and 

(CCI) Committee on Clinical Investigations 
Page 16 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
HIPAA standards.  Access to clinical medical records used for the trial is strictly limited in accordance 
with hospital policies.  Data abstracted from medical records will be managed in compliance with 
GCP clinical research best practices; both hard copy and electronic data collected for the 
investigation will be stored in restricted access files in the TI Clinical Research Offices and in 
electronic files and folders behind hospital firewalls. Data included in analytic files will be de-identified 
and the key linking data to patient identity will be maintained by the investigators in a separated 
restricted access location. 
 
B9 
Multi-Site Studies 
Is the BIDMC the coordinating site?       
 Yes    
 No  
Is the BIDMC PI the lead investigator of the multi-site study?     
 Yes    
 No  
 
B10 
Dissemination of Research Results 
The study investigators will inform participants directly of the results of their individual treatment 
response (responder or non-responder) to IL-2. 
 

(CCI) Committee on Clinical Investigations 
Page 17 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
 
 
Study ID Card  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PATIENT SAFETY CARD – PLEASE KEEP IN YOUR WALLET  
I am participant in a clinical research study. Study treatment is 4 
weeks daily low dose subcutaneous Proleukin® (recombinant IL-2), 
manufactured by Prometheus Laboratories.  
Name: __________________________________ DOB: ________________  
Address: ___________________________   Phone: ___________________  
Emergency Contact (1): ________________ Phone: ___________________  
Emergency Contact (2): ________________ Phone: ___________________  
Liver Transplant Patient – Beth Israel Deaconess Medical Center (BIDMC) 
PATIENT SAFETY CARD – PLEASE KEEP IN YOUR WALLET  
Study Treatment and Investigator Contact 
Proleukin® Dose: 0.3x106 units/m2 SC daily (duration 4 weeks)  
Physician Principal Investigator: Michael Curry, MD, BIDMC 
Transplant Institute. 
You can contact him or an on call Transplant Nurse Coordinator 24/7  
617-632-9700. 
Study IRB Reference #: __2016P-000086_  
Clinicaltrials.gov NCT# __NCT 02739412__ 

(CCI) Committee on Clinical Investigations 
Page 18 of 18 
 
Study Description – Part B 
CCI Form:  9-2015 
 
PI Revision Date:   9.7.2017 
 
Calendar for Medication Recording