Clinical trial NCT01749540
Conditions: B-Thalassemia
Sponsor: Acceleron Pharma, Inc., a wholly-owned subsidiary of Merck & Co., Inc., Rahway, NJ USA
Start date: 2013-02-28; Completion date: 2015-11-11
Enrollment: 64 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to evaluate the efficacy, safety, and pharmacokinetics, of ascending doses of luspatercept in participants with β-thalassemia.

The primary objective of this study is to evaluate erythroid response, defined as:

1. a hemoglobin increase of ≥ 1.5 g/dL from baseline for ≥ 14 days (in the absence of red blood cell \[RBC\] transfusions) in non-transfusion dependent participants, or
2. a ≥ 20% reduction in RBC transfusion burden compared to pretreatment in transfusion dependent participants.

Detailed description:

Arms: arm 1: Participants receive luspatercept 0.2 mg/kg as a subcutaneous (SC) injection every 3 weeks (Q3W) on Day 1 of each cycle for up to 5 cycles (each cycle length = 21 days). arm 2: Participants receive luspatercept 0.4 mg/kg as an SC injection Q3W on Day 1 of each cycle for up to 5 cycles (each cycle length = 21 days). arm 3: Participants receive luspatercept 0.6 mg/kg as an SC injection Q3W on Day 1 of each cycle for up to 5 cycles (each cycle length = 21 days). arm 4: Participants receive luspatercept 0.8 mg/kg as an SC injection Q3W on Day 1 of each cycle for up to 5 cycles (each cycle length = 21 days). arm 5: Participants receive luspatercept 1.0 mg/kg as an SC injection Q3W on Day 1 of each cycle for up to 5 cycles (each cycle length = 21 days). arm 6: Participants receive luspatercept 1.25 mg/kg as an SC injection Q3W on Day 1 of each cycle for up to 5 cycles (each cycle length = 21 days). arm 7: Participants receive an initial dose of luspatercept 0.8 mg/kg as an SC injection on Day 1 of Cycle 1 (Cycle length = 21 days). For each subsequent cycle (up to 5 cycles), the dose was titrated up to a maximum dose of 1.25 mg/kg based on the safety review team (SRT) recommendations.
Interventions: intervention 1: luspatercept
Intervention descriptions: intervention 1: subcutaneous injection

Protocol documents:
Prot_000.pdf: 
Official Protocol Title: 
NCT number: 
Document Date: 
A Phase 2, Open-Label, Ascending Dose Study to Evaluate the 
Effects of ACE-536 in Patients with β-Thalassemia
07-Nov-2014
NCT01749540


Protocol A536-04  
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
          CONFIDENTIAL 
Revision Level: 04 
 
CLINICAL STUDY PROTOCOL 
 
A Phase 2, Open-Label, Ascending Dose Study to Evaluate the Effects 
of ACE-536 in Patients with β-Thalassemia  
 
 
    INVESTIGATIONAL PRODUCT:  
 
Luspatercept (ACE-536) 
 
 
    PROTOCOL NUMBER:  
 
 
 
 
A536-04 
 
 
    EudraCT NUMBER:   
 
 
 
 
 
2012-002499-15 
 
 
SPONSOR: 
Acceleron Pharma Inc. 
128 Sidney Street 
Cambridge, MA 02139 USA 
 
Tel: 
 
Fax: 
 
 
MEDICAL MONITOR: 
 
 
 
 
 
 
 
 
 
 
 
 
Vice President, Medical Research 
 
 
 
ORIGINAL PROTOCOL DATE:  
 
 
06-Aug-2012 
 
AMENDMENT 01 DATE:  
07-Sep-2012 
AMENDMENT 02 DATE: 
09-Apr-2013 
AMENDMENT 03 DATE: 
27-Nov-2013 
AMENDMENT 04 DATE: 
 
 
 
 
07-Nov-2014 
 
 
 
 
 
Confidentiality Statement 
This confidential information in this document is provided to you as an investigator or consultant for review by you, your staff, 
and the applicable Independent Ethics Committee (IEC).  Your acceptance of this document constitutes agreement that you will 
not disclose the information contained herein to others without written authorization from the sponsor. 
 
 
PPD
PPD
PPD






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SITE PRINCIPAL INVESTIGATOR SIGNATURE PAGE 
Signature: 
 
 
 
Date: 
 
Name (print): 
 
 
 
 
 
DD/MMM/YYYY 
 
 
 
 
Institution Name and Address: 
 
 
 
 
 
 
 
 
 
 
 
By my signature I have read the protocol and agree to personally supervise and conduct the study 
as outlined in the protocol.  The study will be conducted in accordance to current United States 
Food and Drug Administration (FDA) regulations, International Conference of Harmonization 
(ICH) Guidelines, Good Clinical Practices (GCP), the Declaration of Helsinki, and local ethical 
and legal requirements. 


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PROCEDURES IN CASE OF EMERGENCY 
Table 1: 
Emergency Contact Information 
Role in Study 
Name 
Contact Information 
Acceleron Medical Monitor 
 
 
Acceleron Pharma Inc. 
128 Sidney Street  
Cambridge, MA 02139 USA 
 
 
 
 
Chiltern Medical Advisor 
 
Chiltern 
171 Bath Road 
Slough, Berkshire, SL1 4AA, 
UK 
 
 
 
  
Pharmacovigilance 
Chiltern 
 
 
 
PPD
PPD
PPD
PPD
PPD


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1. 
PROTOCOL SYNOPSIS 
Name of Sponsor/Company:  
Acceleron Pharma Inc. 
 
128 Sidney Street 
 
Cambridge, MA 02139 USA 
Name of Investigational Product: Luspatercept (ACE-536) 
Name of Active Ingredient:   
ACE-536 is a recombinant fusion protein consisting of a modified form of the extracellular 
domain (ECD) of the human activin receptor IIB (ActRIIB) linked to the human IgG1 Fc domain. 
Title of Study:  
A phase 2, open-label, ascending dose study to evaluate the effects of ACE-536 in patients with 
β-thalassemia  
Study Centers: up to 15  
Phase of Development: 2 
Objectives: 
 Primary:  
 To evaluate the proportion of β-thalassemia patients who have an erythroid 
response, defined as: 1) a hemoglobin increase of ≥ 1.5 g/dL from baseline for 
≥ 14 days (in the absence of red blood cell [RBC] transfusions) in non-transfusion 
dependent patients, or 2) ≥ 20% reduction in RBC transfusion burden compared to 
pretreatment in transfusion dependent patients  
 Secondary: 
 To evaluate safety and tolerability of ACE-536 
 To evaluate the proportion of transfusion dependent patients who have ≥ 50% 
reduction of RBC transfusion burden compared to pretreatment  
 To evaluate the time to erythroid response and duration of erythroid response 
 To evaluate the change in hemoglobin level in non-transfusion dependent patients 
 To evaluate the change in pre-transfusion hemoglobin levels in transfusion 
dependent patients  
 To evaluate changes in biomarkers of erythropoiesis, hemolysis, iron metabolism 
and bone metabolism  
 To examine the pharmacokinetic (PK) profile of ACE-536 in patients with 
β-thalassemia  


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 Exploratory: 
 To evaluate biomarkers related to the TGF- superfamily 
 To evaluate self-reported quality of life using tools including but not limited to the 
FACT-An and SF-36 questionnaires (expansion cohort only)  
 To evaluate change in spleen size by MRI 
 To evaluate change in bone mineral density (BMD) by DXA 
 To evaluate change in extramedullary hematopoiesis (EMH) mass size by MRI  
 To evaluate change in leg ulcer size 
 To evaluate change in the 6-minute walk test (6MWT) distance in NTD patients 
Methodology:  
This is a phase 2, open-label, ascending dose study to evaluate the effects of ACE-536 in patients 
with β-thalassemia.  Patients who meet the study eligibility criteria will be enrolled within 28 days 
of screening.  Patients in all cohorts will receive ACE-536, administered subcutaneously (SC), 
every 3 weeks for up to 5 cycles.  ACE-536 cannot be administered after study day 85 (± 2 days).  
Dose delay(s) and dose reduction(s) may be required for individual patients as outlined in the 
Patient Dose Modification Rules (Section 10.8.1).   
Each dose escalation cohort will consist of up to 6 patients.  The dose level of ACE-536 for the 
first cohort will be 0.2 mg/kg and the dose level(s) for subsequent cohort(s) will follow a modified 
Fibonacci dose escalation scheme (ie, maximum dose of 0.4 mg/kg for cohort 2, 0.6 mg/kg for 
cohort 3, 0.8 mg/kg for cohort 4, 1.0 mg/kg for cohort 5, etc.).  Once a minimum of 3 patients in a 
cohort have completed Study Day 29, a Safety Review Team (SRT) will review preliminary safety 
and hematologic response data and make recommendations to the Sponsor regarding whether or 
not to enroll an additional cohort at a higher or lower dose, complete enrollment of 6 patients at 
the current dose level, and/or proceed to the expansion cohort.   
The expansion cohort (n=30) will be treated with ACE-536 at a starting dose level not to exceed 
the maximum dose level for the previous cohorts.  The expansion cohort will include a minimum 
of 12 patients who are non-transfusion dependent and a minimum of 12 patients who are 
transfusion dependent (including a minimum of 6 thalassemia major patients with onset of regular 
transfusions before age 4 years, and a minimum of 6 patients with onset of regular transfusions 
after age 4 years).  For subsequent cycles in the expansion cohort, a patient’s dose level may be 
titrated from the previous dose administered for that patient.  Patients in the expansion cohort only 
may have their dose level titrated up or down as required per protocol to meet dose titration and 
modification rules (see Section 10.8).  The maximum dose level given to a patient will not exceed 
the maximum dose level evaluated in the dose escalation cohorts.  Patients will be treated with up 
to 5 doses of ACE-536. 
Each TD patient in the expansion cohort will have a defined “pre-transfusion hemoglobin 
threshold” which will be calculated based on transfusion history and will be used for determining 
when to transfuse during the study.  The baseline pre-transfusion hemoglobin threshold will be the 
mean of all documented pre-transfusion hemoglobin values during the 12 weeks prior to C1D1.  
During treatment, if the pre-transfusion hemoglobin value is increased by ≥ 1 g/dL compared to 


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the baseline pre-transfusion hemoglobin threshold for that patient, transfusion should be delayed 
by a minimum of 7 days and/or the number of units transfused should be reduced by 1 or more 
RBC units.  Patients may be transfused at the investigator’s discretion for symptoms related to 
anemia or other requirements (e.g., infection).  
Cohort
a 
ACE-536 Dose Level
b  
(mg/kg) 
Number of Patients 
1 
0.2 
3-6 
2 
0.4 
3-6 
3 
0.6 
3-6 
4 
0.8 
3-6 
5 
1.0 
3-6 
6 
1.25 
3-6 
7 
1.5 
3-6 
Expansion 
TBD 
30c 
Planned Total (All Cohorts):                
Approximately 51-72 
a  Cohort escalation is based on SRT review and recommendation to enroll additional cohorts and/or the 
expansion cohort. 
b  The ACE-536 Dose Level for cohort 1 is 0.2 mg/kg.  The dose level indicated for all subsequent cohorts 
is the maximum dose level that can be recommended by the SRT for escalation per the modified 
Fibonacci dose escalation scheme.  
c  The expansion cohort will consist of 30 patients, including a minimum of 12 patients who are non-
transfusion dependent and a minimum of 12 patients who are transfusion dependent (including a 
minimum of 6 thalassemia major patients with onset of regular transfusions before age 4 years, and a 
minimum of 6 patients with onset of regular transfusions after age 4 years).   
Maximum Number of Patients (Planned): 51-72 patients 
Duration of Treatment: The total duration of participation for a patient is approximately 
24 weeks (4 week screening period, 12 week treatment period, and 8 week follow up period).  If a 
patient has a positive anti-drug antibody (ADA) result at the last visit, the patient may be asked to 
return for additional ADA testing every three months, until a negative result is obtained, or the 
result is considered to be stabilized. 
Diagnosis and Main Criteria for Eligibility 
Inclusion Criteria: 
1. 
Men or women  18 years of age.  
2. 
For the dose escalation phase of the study: documented diagnosis of β-thalassemia intermedia 
(transfusion dependent patients must not have begun regular transfusions at age < 4.0 years).  
For the expansion cohort: documented diagnosis of β-thalassemia (including β-thalassemia 
major or β-thalassemia intermedia). 
3. 
Prior splenectomy or spleen size < 18 cm in the longest diameter by abdominal ultrasound 
(dose escalation cohorts only). 


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4. Anemia, defined as: 
a. Mean hemoglobin concentration < 10.0 g/dL of 2 measurements (one performed 
within one day prior to Cycle 1 Day 1 and the other performed during the screening 
period [Day -28 to Day -1]) in non-transfusion dependent patients, defined as 
having received < 4 units of RBCs within 8 weeks prior to Cycle 1 Day 1, OR 
b. Transfusion dependent, defined as requiring ≥ 4 units of RBCs every 8 weeks 
(confirmed over 6 months prior to Cycle 1 Day 1).  
5. 
Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) < 3 x upper limit of 
normal (ULN). 
6. 
Serum creatinine ≤ 1.5 x ULN. 
7. 
Females of child bearing potential (defined as sexually mature women who have not 
undergone hysterectomy or bilateral oophorectomy, or are not naturally postmenopausal 
≥ 24 consecutive months) must have negative urine or blood pregnancy test prior to 
enrollment and use adequate birth control methods (abstinence, oral contraceptives, barrier 
method with spermicide, or surgical sterilization) during study participation and for 12 weeks 
following the last dose of ACE-536.  Males must agree to use a latex condom during any 
sexual contact with females of child-bearing potential during study participation and for 12 
weeks following the last dose of ACE-536, even if he has undergone a successful vasectomy.  
Patients must be counseled concerning measures to be used to prevent pregnancy and 
potential toxicities prior to the first dose of ACE-536. 
8. 
Patients are able to adhere to the study visit schedule, understand and comply with all 
protocol requirements. 
9. 
Understand and able to provide written informed consent. 
Exclusion Criteria: 
1. Any clinically significant pulmonary (including pulmonary hypertension), 
cardiovascular, endocrine, neurologic, hepatic, gastrointestinal, infectious, 
immunological (including clinically significant allo- or auto-immunization) or 
genitourinary disease considered by the investigator as not adequately controlled prior to 
Cycle 1 Day 1.  
2. Folate deficiency. 
3. Symptomatic splenomegaly. 
4. Known positive for human immunodeficiency virus (HIV), active infectious hepatitis B 
(HBV), or active infectious hepatitis C (HCV). 
5. Known history of thromboembolic events ≥ grade 3 according to the National Cancer 
Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) v.4.0 
(current active minor version). 
6. Ejection fraction < 50% by echocardiogram, MUGA or cardiac MRI. 
7. Uncontrolled hypertension defined as systolic blood pressure (BP) ≥ 150 mm Hg or 


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diastolic BP ≥ 95 mm Hg. 
8. Heart failure class 3 or higher (New York Heart Association, NYHA, Appendix 1). 
9. QTc > 450 msec on screening ECG. 
10. Platelet count < 100 x109/L or > 1,000 x109/L. 
11. Proteinuria ≥ Grade 2. 
12. Any active infection requiring parenteral antibiotic therapy within 28 days prior to 
Cycle 1 Day 1 or oral antibiotics within 14 days of Cycle 1 Day 1.  
13. Treatment with another investigational drug or device, or approved therapy for 
investigational use ≤ 28 days prior to Cycle 1 Day 1, or if the half-life of the previous 
investigational product is known, within 5 times the half-life prior to Cycle 1 Day 1, 
whichever is longer.  
14. Transfusion event within 7 days prior to Cycle 1 Day 1. 
15. Patients receiving or planning to receive hydroxyurea treatment.  Patients must not have 
had hydroxyurea within 90 days of Cycle 1 Day 1.    
16. Splenectomy within 56 days prior to Cycle 1 Day 1. 
17. Major surgery (except splenectomy) within 28 days prior to Cycle 1 Day 1.  Patients 
must have completely recovered from any previous surgery prior to Cycle 1 Day 1. 
18. Iron chelation therapy if initiated within 56 days prior to Cycle 1 Day 1. 
19. Cytotoxic agents, systemic corticosteroids, immunosuppressants, or anticoagulant 
therapy such as warfarin or heparin within 28 days prior to Cycle 1 Day 1 (prophylactic 
aspirin up to 100 mg/day is permitted). 
20. Pregnant or lactating females.  
21. History of severe allergic or anaphylactic reactions or hypersensitivity to recombinant 
proteins or excipients in the investigational drug. 
22. Prior treatment with sotatercept (ACE-011) or ACE-536. 
Investigational Product and Mode of Administration: 
ACE-536 drug product is provided as a sterile, liquid formulation at a nominal concentration of 
50 mg/mL in Tris-buffered saline, pH 7.2 ± 0.5, contained in type I, 13-mm borosilicate glass vials 
(stoppered and crimp-sealed).  Each single-use vial contains 0.5 mL (25 mg) of ACE-536 solution 
for injection. 
ACE-536 will be administered by SC injection on Day 1 of each cycle. 
Safety Evaluation and Dose Escalation: 
Safety will be evaluated by the Safety Review Team (SRT), which is comprised at minimum of 
the coordinating investigator, medical monitor, and an independent hematologist.  The role of the 
SRT will be described in greater detail in the SRT Charter.  The SRT will review safety data when 
a minimum of 3 patients in a cohort have completed Study Day 29, including dose-limiting 


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toxicities (DLTs), adverse events (AEs), serious adverse events (SAEs), laboratory results 
(including hematology and chemistry), vital signs, and erythroid response data.  The SRT may 
make one or more of the following recommendations to the sponsor: 
 
Enroll 3-6 patients in an additional cohort at a higher or lower dose level, not to exceed 
modified Fibonacci dose escalation scheme (see Section 9.1); enrollment in current cohort 
may be closed after a minimum of 3 patients. 
 
Proceed to the expansion cohort, with a recommended starting dose level for TD and NTD 
patients, not to exceed the maximum dose level evaluated in the dose escalation cohorts. 
 
Postpone a decision pending collection of additional data from the current cohort. 
 
Recommend that no further cohorts should be enrolled in the study. 
A DLT, using the current active minor version of the National Cancer Institute Common Toxicity 
Criteria for Adverse Events, version 4.0 (NCI-CTCAE v4.0), is defined as any of the following 
toxicities at any dose level occurring within 28 days of the first administered dose: 
 Treatment related serious adverse event (SAE) of ≥ Grade 3 
 Treatment related non-hematologic adverse event (AE) of ≥ Grade 3 
 Treatment related hematologic AE of ≥ Grade 4 
Safety review team recommendations to escalate or reduce the dose level of ACE-536 in a 
subsequent cohort, or complete enrollment of 6 patients in the current cohort, will be based in part 
upon the following criteria: 
 If a DLT occurs in ≤ 1 patient in a cohort within 28 days following the initial dose of 
ACE-536, dose escalation may occur; 
 If a DLT occurs in ≥ 2 patients in a cohort within 28 days of the initial dose, the dose level 
for the next cohort (if any) should not be escalated; a lower dose may be recommended. 
 If a hemoglobin increase of ≥ 2.0 g/dL (confirmed within 48 to 72 hours and not 
attributable to RBC transfusion) occurs in ≥ 2 patients in a cohort within 28 days of the 
initial dose, the dose level for the next cohort (if any) should not be escalated; a lower dose 
may be recommended. 


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Criteria for Evaluation 
 Safety: All patients will be assessed for safety by monitoring adverse events, clinical 
laboratory tests, vital signs, ECG, and physical examination.   
 Efficacy: Patients will be assessed for erythroid response for up to 20 weeks following 
initiation of treatment.  Erythroid response endpoints will be determined by monitoring 
hematological laboratory values and RBC transfusions. 
Secondary efficacy endpoints will be assessed by examining other hematology, 
erythropoiesis, iron metabolism, and bone metabolism parameters, as well as MRI scan for 
liver iron content (LIC). 
o Erythropoiesis parameters include serum erythropoietin levels, hemoglobin analysis 
(electrophoresis, globin chain RNA), reticulocytes, nucleated RBCs, and RBC 
morphology 
o Hemolysis parameters include haptoglobin, indirect bilirubin, and lactate 
dehydrogenase (LDH) 
o Iron metabolism parameters include serum iron, total iron binding capacity (TIBC), 
transferrin, soluble transferrin receptor, ferritin, non-transferrin bound iron (NTBI), 
hepcidin, and LIC by MRI 
o Bone metabolism parameters include bone specific alkaline phosphatase (BSAP) 
and C-telopeptide of type I collagen (CTX) 
 Exploratory endpoints will include:  
o Biomarkers related to the TGF- superfamily 
o Self-reported quality of life measures including but not limited to the FACT-An 
and SF-36 questionnaires (expansion cohort only) 
o Spleen size by MRI 
o Bone mineral density (BMD) by DXA 
o Extramedullary hematopoiesis (EMH) mass size by MRI 
o Leg ulcer size 
o 6-minute walk test (6MWT) distance in NTD patients 
Statistical methods  
Sample Size Calculation: 
There is no formal sample size calculation for the dose escalation portion of the study.  A sample 
size of 30 evaluable patients in the expansion cohort will provide approximately 87% power with 
1-sided significance level of 0.05 to differentiate an erythroid response rate of 30% from a 
minimal erythroid response rate of 10%. 
Analysis Populations: 
For all analysis populations, patients will be analyzed according to assigned cohort. 


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The Intent-to-Treat (ITT) Population: All patients enrolled in the study. 
Efficacy Evaluable (EE) Population: All patients administered at least one dose of ACE-536, 
and 1) at least 2 hemoglobin values ≥ 14 days apart post-treatment, not influenced by transfusion, 
in non-transfusion dependent patients; OR, 2) at least 12 weeks of transfusion frequency data prior 
to Cycle 1 Day 1 and following Cycle 1 Day 1 in transfusion dependent patients. 
Safety Population: All patients who received at least 1 dose of ACE-536. 
Pharmacokinetics Population: All patients who have received at least 1 dose of ACE-536 and 
have sufficient pharmacokinetic (PK) samples collected and assayed for PK analysis. 
Efficacy/Pharmacodynamic Effects Analysis:  
The primary efficacy endpoint is defined as the proportion of β-thalassemia patients who have an 
erythroid response, defined as 1) a hemoglobin increase of ≥ 1.5 g/dL from baseline for ≥ 14 days 
(in the absence of RBC transfusions) in non-transfusion dependent patients or 2) ≥ 20% reduction 
in RBC transfusion burden compared to pretreatment in transfusion dependent patients. 
Transfusion burden will be defined as the ratio of RBC transfusion units (or mLs) transfused 
during an interval to the duration (days) of that interval, where the interval may be during the 
pretreatment period or the treatment plus follow-up period.  The interval during the pretreatment 
period will be defined as the 12 weeks prior to Cycle 1 Day 1.  An interval during the treatment 
plus follow-up period will be defined as any 12-week interval after Cycle 1 Day 1. 
The erythroid response will be summarized using both a point estimate and its exact 95% 
confidence interval based on binomial distribution.  The primary efficacy analysis will be 
performed using the EE population. 
No direct comparison testing with concurrent or historical controls will be performed.   
The secondary efficacy/pharmacodynamic analysis will be performed using the ITT and EE 
populations and will include: 
 Erythroid response, as defined above for primary endpoint, performed using the ITT 
population.  
 For non-transfusion dependent patients, the proportion of patients with an increase of 
≥ 1.0 g/dL from pretreatment hemoglobin maintained ≥ 8 weeks. 
 For non-transfusion dependent patients, the proportion of patients with an increase of 
≥ 1.5 g/dL from pretreatment hemoglobin maintained ≥ 8 weeks. 
 For transfusion dependent patients, the proportion of patients who have ≥ 50% reduction in 
RBC transfusion burden compared to pretreatment using 12-week intervals. 
 For transfusion dependent patients, the proportion of patients who have ≥ 50% reduction in 
RBC transfusion burden compared to pretreatment using 8-week intervals.  
 For transfusion dependent patients, the proportion of patients who have no RBC 
transfusions ≥ 8 weeks. 
 Time to erythroid response and duration of erythroid response 
 Change from pretreatment in transfusion burden in transfusion dependent patients 


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 Change in hemoglobin level in non-transfusion dependent patients 
 Change in pre-transfusion hemoglobin levels in transfusion dependent patients  
 Change from baseline in erythropoiesis parameters including serum erythropoietin levels, 
hemoglobin analysis (electrophoresis, globin chain RNA), reticulocytes, and nucleated 
RBCs 
 Change from baseline in hemolysis parameters include haptoglobin, indirect bilirubin, and 
lactate dehydrogenase (LDH) 
 Change from baseline in iron metabolism parameters include serum iron, total iron binding 
capacity (TIBC), transferrin, calculated transferrin saturation, soluble transferrin receptor, 
ferritin, non-transferrin bound iron (NTBI), hepcidin, and LIC by MRI 
 Change from baseline in bone metabolism parameters include bone specific alkaline 
phosphatase (BSAP) and C-telopeptide of type I collagen (CTX)  
The exploratory endpoints will include evaluation of biomarkers related to the TGF- 
superfamily, quality of life, change in spleen size (in subset of patients with no prior splenectomy), 
change in BMD, change in EMH mass size, change in leg ulcer size, and change in the 6MWT 
distance in NTD patients. 
All binary endpoints will be summarized using both a point estimate and its exact confidence 
interval based on the binomial distribution.  The time-to-event type secondary endpoints will be 
analyzed using Kaplan-Meier method to estimate the survival curve and median time to event and 
95% confidence interval. 
Safety analysis: To assess clinical safety, adverse events, vital sign measurements, and clinical 
laboratory information will be summarized.  Adverse Events will be coded using the Medical 
Dictionary for Regulatory Activities (MedDRA).  Changes from baseline for clinical laboratory 
values and vital signs will be summarized over time.  Descriptive statistics and shift tables will be 
generated as appropriate. 
Pharmacokinetics analysis: Non-compartmental PK parameters for ACE-536, such as maximum 
plasma concentration (Cmax), time to maximum plasma concentration (tmax), and area under the 
concentration/time curve (AUC), will be estimated.  Dose proportionality may be assessed using 
the exposure data (e.g., Cmax, AUC) after the first dose if sufficient dose levels are studied.  
Descriptive statistics will be provided for serum concentrations and PK parameters.  The 
relationship between ACE-536 exposure and response (i.e., safety, efficacy, and biomarkers) may 
be explored, if appropriate. 
Anti-drug antibody analysis: The results of anti-drug and neutralizing antibody testing for 
ACE-536 and human ActRIIB protein versus time will be presented.  Exploratory analysis will be 
performed on the potential effect of anti-drug antibodies on ACE-536 PK and drug exposure if 
anti-drug antibody tests are deemed positive. 


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2. 
SCHEDULE OF EVENTS 
 
 
 
 
Screen 
Treatment Period 
Follow up period 
Cycle 1 
Cycle 2 
Cycle 3 
Cycle 4 
Cycle 5 
EOT12 
EOS13 
C1D12 
C1D8 
C1D11 
C1D15 
C2D12,15 
C2D8 
C2D15 
C3D12,15 
C3D8 
C3D15 
C4D12,15 
C4D8 
C4D15 
C5D12,15 
C5D8 
C5D15 
Day 
113 
(± 7d) 
Day 
141 
(± 7d) 
Day -28 to 
-1 
Day 
1 
Day 
8 
(± 1d) 
Day 
11 
(± 1d) 
Day 
15 
(± 1d) 
Day14 
22 
(± 2d) 
Day 
29 
(± 2d) 
Day 
36 
(± 2d) 
Day14 
43 
(± 2d) 
Day 
50 
(± 2d) 
Day 
57 
(± 2d) 
Day14 
64 
(± 2d) 
Day 
71 
(± 2d) 
Day 
78 
(± 2d) 
Day14 
85 
(± 2d) 
Day 
92 
(± 2d) 
Day 
99 
(± 2d) 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion  
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
QoL Questionnaires19 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X20 
X 
Physical examination 
X 
X23 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X23 
X23 
Vital signs1  
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
ECG (12 lead) 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
MRI (Liver Iron Content)17 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
MRI for EMH masses and 
spleen24 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
DXA for BMD of total body, 
lumbar spine, and total hip24 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Leg Ulcer Assessment25 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Abdominal Ultrasound21 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
ECHO, MUGA or cardiac 
MRI 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6MWT24 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Serum iron studies4 
X 
X 
X 
 
X 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Serum folate and B12 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Erythropoietin levels  
X 
X 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
X 
Hematology5 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Peripheral blood smear 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
Serum chemistry6 
X 
X 
 
 
X 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Urinalysis and Urine 
Chemistry7 
X 
X 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
X 
Anti-drug antibody16  
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
X16 
PK collection 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
PD Biomarkers8 
 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Hemoglobin electrophoresis 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
Globin mRNA sample 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
Bone Biomarkers9 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
Pregnancy test/menstrual 
history10 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X22 
Evaluate transfusion 
frequency/volume11 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Concomitant medications and 
AEs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Administer ACE-53618  
 
X 
 
 
 
X14 
 
 
X14 
 
 
X14 
 
 
X14 
 
 
 
 


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1  
Vital signs: Weight, heart rate, systolic and diastolic blood pressure, respiration rate, and temperature (measured in degrees Celsius). Height is measured only at Screening.  If at any visit 
the systolic blood pressure is ≥ 150 mmHg, the diastolic blood pressure is ≥ 95 mmHg, and/or the absolute increase in either measure from baseline is ≥ 20 mmHg, perform one repeat of 
the blood pressure assessment after a minimum of 15 minutes.   
2  Study procedures must be done prior to administration of study drug.    
3  Screening MRI for Liver Iron Content, MRI of the chest and abdomen for EMH masses and spleen (expansion cohort only), BMD by DXA (expansion cohort only), abdominal ultrasound of the 
spleen (if no prior splenectomy; dose escalation cohorts only) and ECHO, MUGA or cardiac MRI can be performed up to 56 days prior to C1D1.  If performed as part of standard of care, it does 
not need to be repeated. 
4  Iron Studies: Serum iron, TIBC, transferrin, soluble transferrin receptor, ferritin, and NTBI. 
5  
Hematology: RBC, WBC with differential, hemoglobin, hematocrit, haptoglobin (optional), reticulocyte count, platelet count, MCV, MCH, MCHC, RDW, and nRBCs.  On dosing days, 
hematology values are to be drawn and resulted (up to 1 day) prior to C1D1 (see Section 10.8.1 Patient Dose Modification Rules).  Historical hemoglobin values will be collected for 12 
weeks prior to C1D1.  Historical transfusion history will be collected for 12 months prior to Cycle 1 Day 1.  Baseline hemoglobin will be the average of two measurements; one measure 
performed within one day prior to Cycle 1 Day 1 and the other performed during the screening period (Day -28 to Day -1).  Note: For any RBC transfusions received during the study, 
collect hemoglobin value just prior to transfusion.  
6  Chemistry: Sodium, potassium, chloride, carbon dioxide/bicarbonate (optional), AST, ALT, lactate dehydrogenase (LDH), total bilirubin, indirect bilirubin, alkaline phosphatase, blood 
urea nitrogen (BUN)/urea, creatinine, GGT, calcium, phosphorus, glucose, amylase, lipase, total protein, albumin, and uric acid.  
7  Urinalysis by dipstick analysis: pH, specific gravity, protein, glucose, ketones, blood, leukocyte esterase, and nitrite, with microscopic examination if indicated.  Microalbumin and creatinine 
will be performed by the central lab. 
8   PD Biomarkers: Hepcidin, GDF15 and others to be determined. 
9   Bone Biomarkers: BSAP and CTX. 
10    Pregnancy test (urine or serum) and menstrual history is required prior to C1D1 for female patients of child bearing potential only. 
11  Transfusion history will be collected for 12 months prior to C1D1. 
12  End of Treatment (EOT): Should be performed 28 days (± 7 days) after the last dose of ACE-536.  Patients who discontinue treatment early should complete the end of treatment visit 
at the time of discontinuation and complete the Day 141 follow up visit 28 days (± 7 days) after the EOT visit. 
13  End of Study (EOS): Should be performed 28 days (± 7 days) after the Day 113/EOT visit (56 days after the last dose of ACE-536).  
14  Day 85 ± 2 days is the last possible study day that ACE-536 may be administered, regardless of the cycle. 
15  If a dose delay is required per the dose modification rules the patient will not be dosed. The patient will return weekly to assess hematology results and adverse events until the patient is eligible 
to administer the next dose of ACE-536. 
16  
 If the patient has a positive ADA result at their last assessment, the patient may be asked to return approximately every three months for additional testing until a negative result is obtained or the 
result is considered stabilized. 
17   MRI for liver iron content will be performed at selected sites. 
18 For the first dose of ACE-536, dosing should occur after a minimum of 7 days post-transfusion; subsequent doses should not be given within 24 hours of transfusion or planned transfusion. 
19  Administration of quality of life questionnaires, including but not limited to FACT-An and SF-36 is required for patients in the expansion cohort only. 
20  QoL questionnaires including but not limited to FACT-An and SF-36 should be completed at the EOT visit only for expansion cohort patients who discontinue treatment early. 
21  Abdominal ultrasound of the spleen is required for patients with no prior splenectomy in the dose escalation cohorts only.  Patients in these cohorts with clinical signs of a change in spleen size or 
abnormality should have an abdominal ultrasound as needed throughout study. 
22  Pregnancy test is not required at Day 141/EOS. 
23  Physical exam should include an optional evaluation of gonadal size in males at C1D1, EOT, and EOS visits only. 
24  MRI of the chest and abdomen for EMH masses and spleen, DXA for BMD and 6MWT for NTD patients will be performed in the expansion cohort patients only, at selected sites. 
25  Patients with leg ulcers should have regular assessment of the leg ulcer(s) throughout the study.  Photographs of the leg ulcer(s) should be obtained to document any changes in leg ulcer(s) size. 


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3. 
TABLE OF CONTENTS 
1. 
PROTOCOL SYNOPSIS .............................................................................................6 
2. 
SCHEDULE OF EVENTS .........................................................................................15 
3. 
TABLE OF CONTENTS ...........................................................................................17 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................21 
5. 
ETHICS ......................................................................................................................24 
5.1. 
Institutional Review Board .........................................................................................24 
5.2. 
Ethical Conduct of the Study ......................................................................................24 
5.3. 
Patient Information and Consent ................................................................................24 
5.4. 
Patient Data Protection ...............................................................................................24 
6. 
INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE .................25 
7. 
INTRODUCTION ......................................................................................................26 
7.1. 
Overview of Target Indication ....................................................................................26 
7.2. 
Summary of Nonclinical Studies ................................................................................27 
7.2.1. 
Pharmacology Studies ................................................................................................27 
7.2.2. 
Toxicology Studies .....................................................................................................29 
7.3. 
Summary of Clinical Experience ................................................................................30 
7.4. 
Potential Risks of Human Use ....................................................................................31 
8. 
TRIAL OBJECTIVES ................................................................................................33 
8.1. 
Primary Objective .......................................................................................................33 
8.2. 
Secondary Objectives .................................................................................................33 
8.3. 
Exploratory Objective .................................................................................................33 
9. 
OVERALL STUDY DESIGN AND PLAN: DESCRIPTION ..................................34 
9.1. 
Study Design ...............................................................................................................34 
9.2. 
Discussion of Study Design ........................................................................................35 
9.3. 
Selection of Study Population ....................................................................................36 
9.3.1. 
Inclusion Criteria ........................................................................................................36 
9.3.2. 
Exclusion Criteria .......................................................................................................37 
9.4. 
Patient Treatment Discontinuation and Withdrawal Criteria .....................................38 
10. 
TREATMENT OF PATIENTS ..................................................................................39 
10.1. 
Selection and Timing of Dose for Each Patient ..........................................................39 


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10.2. 
Concomitant Medications ...........................................................................................39 
10.2.1. 
Other Treatments for β-Thalassemia ..........................................................................39 
10.2.2. 
RBC Transfusions .......................................................................................................39 
10.3. 
Treatment Compliance ................................................................................................40 
10.4. 
Randomization ............................................................................................................40 
10.5. 
Treatments Administered ............................................................................................40 
10.6. 
Dose-Limiting Toxicity Definition .............................................................................40 
10.7. 
Safety Evaluation and Dose Escalation ......................................................................40 
10.8. 
Patient Dose Modification and Titration ....................................................................41 
10.8.1. 
Patient Dose Modification Rules ................................................................................42 
10.8.2. 
Individual Dose Titration Rules for NTD Patients in the Expansion Cohort .............43 
10.8.3. 
Individual Dose Titration Rules for TD Patients in the Expansion Cohort ................43 
10.8.4. 
Other Considerations for Dose Modification, Delay or Discontinuation ...................43 
11. 
STUDY PROCEDURES ............................................................................................44 
11.1. 
Written Informed Consent ..........................................................................................44 
11.2. 
Clinical Laboratory Tests ...........................................................................................44 
11.3. 
Other Safety Assessments ...........................................................................................44 
11.4. 
Pharmacokinetic and Pharmacodynamic Assessments ..............................................45 
11.4.1. 
Pharmacokinetic Assessments ....................................................................................45 
11.4.2. 
Pharmacodynamic Evaluations ...................................................................................45 
12. 
STUDY SCHEDULE .................................................................................................46 
12.1. 
Screening ....................................................................................................................46 
12.2. 
Dosing Days and Interim Visits ..................................................................................47 
12.3. 
Day 85 .........................................................................................................................48 
12.4. 
End of Treatment Visit ...............................................................................................48 
12.5. 
End of Study Visit ......................................................................................................49 
12.6. 
Termination of Study ..................................................................................................49 
13. 
STUDY DRUG MATERIALS AND MANAGEMENT ...........................................50 
13.1. 
Study Drug ..................................................................................................................50 
13.2. 
Study Drug Packaging and Labeling ..........................................................................50 
13.3. 
Study Drug Storage .....................................................................................................50 
13.4. 
Study Drug Preparation ..............................................................................................50 
13.5. 
Study Drug Administration.........................................................................................50 


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13.6. 
Study Drug Accountability .........................................................................................50 
13.7. 
Study Drug Handling and Disposal ............................................................................51 
14. 
ASSESSMENT OF SAFETY .....................................................................................51 
14.1. 
Adverse Event Definitions ..........................................................................................51 
14.2. 
Pregnancy and In Utero Drug Exposure .....................................................................52 
14.3. 
Severity .......................................................................................................................52 
14.4. 
Relationship to Study Drug ........................................................................................53 
14.5. 
Documentation and Methods of Reporting of Adverse Events by Investigator .........53 
14.5.1. 
Documentation of Serious Adverse Events ................................................................54 
14.6. 
Reporting Period and Monitoring of Patients with Adverse Events ...........................54 
14.7. 
Notification about Serious Adverse Events ................................................................54 
14.7.1. 
Safety Reporting to Health Authorities, Independent Ethics Committees, 
Institutional Review Boards, and Investigators ..........................................................55 
15. 
STATISTICS ..............................................................................................................56 
15.1. 
Analysis Populations ..................................................................................................56 
15.2. 
Statistical Plan ............................................................................................................56 
15.2.1. 
Patient Accountability and Demographics .................................................................56 
15.2.2. 
Primary Efficacy Analysis ..........................................................................................56 
15.2.3. 
Secondary Analysis ....................................................................................................57 
15.2.4. 
Pharmacokinetics analysis ..........................................................................................58 
15.2.5. 
Anti-drug antibody analysis ........................................................................................58 
15.3. 
Determination of Sample Size ....................................................................................58 
15.4. 
Interim Analysis ..........................................................................................................58 
15.5. 
Deviation from Original Analysis Plan ......................................................................59 
16. 
SOURCE DOCUMENTATION AND INVESTIGATOR FILES .............................59 
16.1. 
Study Monitoring ........................................................................................................59 
16.2. 
Audits and Inspections ................................................................................................59 
17. 
QUALITY CONTROL AND QUALITY ASSURANCE .........................................60 
17.1. 
Data Quality Control and Quality Assurance .............................................................60 
17.1.1. 
Investigator Responsibility .........................................................................................60 
17.1.2. 
Protocol Modifications ...............................................................................................60 
18. 
CONFIDENTIALITY ................................................................................................61 
19. 
PROTOCOL AMENDMENTS ..................................................................................61 


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20. 
DATA HANDLING AND RECORDKEEPING .......................................................62 
20.1. 
Case Report Form Completion ...................................................................................62 
20.2. 
Retention of Records ..................................................................................................62 
21. 
STUDY FINANCE AND INSURANCE ...................................................................63 
21.1. 
Study Finance .............................................................................................................63 
21.2. 
Insurance .....................................................................................................................63 
22. 
REFERENCES ...........................................................................................................64 
23. 
APPENDICES ............................................................................................................65 
23.1. 
Appendix 1: New York Heart Association Classification - The Stages of 
Heart Failure9 ..............................................................................................................65 
23.2. 
Appendix 2: National Cancer Institute (NCI) Common Terminology Criteria 
for Adverse Events (CTCAE) .....................................................................................66 


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4. 
 LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation 
Definition 
6MWT 
Six minute walk test 
ABPI 
Association of the British Pharmaceutical Industry 
ActRIIB 
Activin receptor IIB 
ADA 
Anti-drug antibody 
AE 
Adverse Event 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase 
AUC 
Area under the concentration-time curve 
BFU-E 
Burst forming units - erythroid 
BMD 
Bone mineral density 
BMP 
Bone morphogenetic protein 
BSAP 
Bone specific alkaline phosphatase 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CXDY 
Cycle X Day Y 
CFU-E 
Colony forming units - erythroid 
CI 
Confidence interval 
Cmax 
Maximum concentration 
CREAT 
Creatinine 
CRF 
Case report form 
CRO 
Contract research organization 
CTX 
C-telopeptide of type I collagen 
DLT 
Dose-limiting toxicity 
DXA 
Dual-energy X-ray absorptiometry 
ECD 
Extracellular domain 
ECHO 
Echocardiogram 
ECG 
Electrocardiogram 
EE 
Efficacy Evaluable 
EMH 
Extramedullary hematopoiesis 
EOS 
End of Study 


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Abbreviation 
Definition 
EPO 
Erythropoietin 
EOT 
End of Treatment 
FACS 
Fluorescence activated cell sorting 
FACT-An 
Functional Assessment of Cancer Therapy-Anemia Scale 
FDA 
United States Food and Drug Administration 
GCP 
Good clinical practices 
GDF 
Growth and differentiation factor 
GGT 
Gamma-glutamyl transpeptidase 
HbA 
Adult hemoglobin 
HbF 
Fetal hemoglobin 
HBV 
Hepatitis B virus 
HCV 
Hepatitis C virus 
HIV 
Human immunodeficiency virus 
ICF 
Informed consent form 
ICH 
International conference on harmonisation 
IEC 
Independent ethics committee 
IgG1 
Immunoglobulin G1 
ITT 
Intent-to-Treat 
IB 
Investigator’s brochure 
KD 
Dissociation constant 
LIC 
Liver iron content 
LDH 
Lactate dehydrogenase 
MedDRA 
Medical Dictionary for Regulatory Activities 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
MRI 
Magnetic resonance imaging 
MUGA 
Multi gated acquisition scan 
NCI-CTCAE 
National Cancer Institute-Common terminology criteria for adverse events 
NYHA 
New York Heart Associate 
nRBCs 
Nucleated red blood cells 
NTBI 
Non-transferrin bound iron  


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Abbreviation 
Definition 
PD 
Pharmacodynamic 
PHI 
Protected health information 
PK 
Pharmacokinetic 
QoL 
Quality of life 
RBC 
Red blood cell 
RDW 
Red blood cell distribution width 
RNA 
Ribonucleic acid 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SC 
Subcutaneous 
SD 
Standard deviation 
SRT 
Safety review team 
SF-36 
Short form (36) health survey 
SUSAR 
Suspected unexpected serious adverse reaction 
T1/2 
Elimination half-life 
TGF-β 
Transforming growth factor beta 
TIBC 
Total iron binding capacity 
Tmax 
Time to maximum concentration 
ULN 
Upper limit of normal 
WBC 
White blood cell 


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5. 
ETHICS 
5.1. 
Institutional Review Board 
The investigator will submit this protocol, any protocol modifications, and the patient Informed 
Consent Form (ICF) to be used in this study to the appropriate IEC for review and approval.  A 
letter confirming IEC approval of the protocol and ICF as well as a statement that the IEC is 
organized and operates according to Good Clinical Practice (GCP) and the applicable laws and 
regulations, must be forwarded to the sponsor prior to the enrollment of patients into the study.  
A copy of the approved ICF will also be forwarded to the sponsor.  Appropriate reports on the 
progress of the study will be made to the IEC and the sponsor by the principal investigator in 
accordance with applicable governmental regulations and in agreement with the policy 
established by the sponsor. 
5.2. 
Ethical Conduct of the Study 
The sponsor and the investigator must comply with all instructions, regulations, and agreements 
in this protocol and in the applicable International Conference on Harmonisation (ICH) and GCP 
guidelines, and must also conduct the study in accordance with local regulations.   
5.3. 
Patient Information and Consent 
Informed written consent is required from each patient prior to any testing under this protocol, 
including screening tests and evaluations.  The ICF, as specified by the study center’s IEC, must 
follow the Protection of Human Patients regulations listed in the Code of Federal Regulations, 
Title 21, Part 50.  The principles of informed consent in the Declaration of Helsinki should be 
implemented in this clinical study and should comply with local and national regulations.  The 
consent forms must be in a language fully comprehensible to the prospective subject. 
Information should be given in both oral and written form whenever possible and deemed 
appropriate by the IEC. 
The background of the proposed study and the benefits and risks of the procedures and study 
must be explained to the patients.  It is the responsibility of the investigator to obtain consent and 
to provide the patient with a copy of the signed and dated ICF.  Confirmation of a patient’s 
informed consent must also be documented in the patient’s medical record prior to any testing 
under this protocol, including screening tests and evaluations. 
All ICFs used in this study must be approved by the appropriate IEC and by the Sponsor or 
designee.  The ICF must not be altered without the prior agreement of the relevant IEC and the 
Sponsor.   
5.4. 
Patient Data Protection 
Prior to any testing under this protocol, including screening tests and evaluations, patients must 
authorize the release and use of protected health information (PHI), as required by local law. 
The patient will not be identified by name in the case report form (CRF) or in any study reports.  
These reports will be used for research purposes only.  The Sponsor, its designee, and various 


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government health agencies may inspect the records of this study.  Every effort will be made to 
keep the patient’s personal medical data confidential. 
6. 
INVESTIGATORS AND STUDY ADMINISTRATIVE 
STRUCTURE 
Acceleron Pharma is the sponsor for this trial.  The sponsor will serve as the medical monitor for 
the study.  The sponsor or designee also will manage the conduct of the trial and provide for 
clinical monitoring, data management, biostatistics, and report writing.  Clinical monitors will 
monitor each study center on a periodic basis and verify source documentation for each patient.  
The sponsor’s pharmacovigilance representative will be responsible for timely reporting of 
Serious Adverse Events (SAEs) to health authorities as required. 


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7. 
INTRODUCTION 
ACE-536 is a recombinant fusion protein consisting of a modified form of the extracellular 
domain (ECD) of the human activin receptor type IIB (ActRIIB) linked to the human IgG1 Fc 
domain.  The ActRIIB receptor and its ligands are members of the TGF-β superfamily, a group 
of proteins involved in the development, differentiation, and/or maturation of various tissues.  No 
species differences have been described in the ligand-receptor interactions among members of 
the TGF-β family as the ligands and receptors are highly conserved across species.1  Thus, 
observations from pharmacology studies of ACE-536 or its murine analog RAP-536 in animal 
models provide significant insight into the potential of ACE-536 to treat human disease. 
Members of the TGF-β family are reported to play a role in red blood cell (RBC) development, 
although the specific ligands and receptors that influence the process of erythropoiesis are not 
completely understood.  In nonclinical experiments, ACE-536 has been shown to bind with high 
affinity to some TGF-β ligands (eg, GDF8, GDF11, BMP6, and activin B), but substantially less, 
or not at all, to others (eg, BMP10 and activin A).  The emerging body of evidence on ACE-536 
suggests that its mechanism of action is distinct from that of erythropoietin (EPO), and involves 
acceleration of the later maturation phase of erythrocyte development. 
The nonclinical pharmacology data using ACE-536/RAP-536 demonstrate that the molecule can 
produce a rapid and robust erythroid response in a variety of settings, and that this response is 
due to its influence on the differentiation of hematopoietic cell types in the later stages of 
erythropoiesis.  In contrast, EPO is active in the early, proliferative stage of erythropoiesis.  
ACE-536 has shown activity in animal models of various conditions in which anemia contributes 
significantly to disease morbidity, including anemia associated with ineffective erythropoiesis.  
In a murine model of β-thalassemia, RAP 536 showed significant improvements in hematologic 
indices (red cell count, hemoglobin, hematocrit, red cell distribution width, and red cell 
morphology).  Additionally, hemolysis was significantly reduced in RAP-536 treated mice, as 
measured by reduction in serum total bilirubin.  Importantly, the mechanism of RAP-536 action 
in β-thalassemia mice was shown to be related to the accelerated maturation of erythroid 
precursor cell populations, specifically, increases in the orthochromatic erythroblasts and 
reticulocytes in the bone marrow and spleen, with concomitant reductions in the earlier-stage 
basophilic erythroblast population. 
7.1. 
Overview of Target Indication 
The target indication for ACE-536 is for the treatment of symptomatic anemia in patients with 
β-thalassemia.  Anemia in β-thalassemia arises as a consequence of three main components, 
(i) ineffective erythropoiesis, (ii) red cell hemolysis, and (iii) abnormal red cell morphology.  
The most important of these is ineffective erythropoiesis, which is characterized by erythroid 
hyperplasia and substantially increased rates of apoptosis of erythrocyte precursors in the bone 
marrow, particularly the polychromatophilic and orthochromatic erythroblasts.2  Apoptotic 
mechanisms are known to play a physiological role in the process of normal erythrocyte 
development; it is possible that these mechanisms are exacerbated in β-thalassemia.3  Ineffective 
erythropoiesis can lead to erythroid expansion in the bone marrow which in turn can lead to bony 
deformities and osteoporosis, making patients with β-thalassemia more susceptible to fractures.  
The acceleration of red blood cell destruction can also result in splenomegaly.4  


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Onset of anemia in β-thalassemia generally occurs between 6 and 24 months of age, 
corresponding to the switch from the γ-chain of fetal hemoglobin (HbF) the β-chains of adult 
hemoglobin (HbA).  The exact prevalence of β-thalassemia in the world population is unknown.  
In the Eastern Mediterranean it is estimated that there are 10,000 cases annually and of these 
cases approximately 9,000 are transfusion dependent.5  According to Orpha.net (Report Series 
2 May 2012) the estimated prevalence of β-thalassemia in Europe is 1-9/1,000,000, with a higher 
prevalence in Mediterranean regions.  
General care for patients with β-thalassemia requires substantial supplemental pediatric care, 
including careful attention to nutrition and monitoring for signs of infection, red cell transfusions 
to prevent the damage of chronic anemia, and iron chelation to prevent the damage of iron 
overload due to ineffective erythropoiesis and regular blood transfusion.  In older patients with 
signs of iron overload, supportive care is typically required to address endocrine insufficiencies, 
metabolic bone disease, and cardiac failure.  Children with β-thalassemia who are periodically 
transfused to maintain a hemoglobin value of 9.5 to 14 g/dL grow and develop normally.6  
However, these patients require lifelong transfusion support, and concomitant iron chelation 
therapy to maintain a reasonable quality of life.  Iron chelation therapy itself, can be associated 
with a number of side-effects, including neurosensory toxicities and retardation of bone.  Iron-
related cardiotoxicity is the most common cause of mortality in patients with β-thalassemia. 
Advances in prenatal diagnosis have reduced incidence rate of β-thalassemia in developed 
countries, but have had little impact on the global incidence rate.7  Treatments that have the 
potential to attenuate ineffective erythropoiesis and correct the anemia that characterize the 
disease could provide significant clinical benefit to patients by reducing the need for periodic 
blood transfusions and associated iron chelation therapy.  ACE-536 has the potential to provide 
benefit in a variety of conditions in which ineffective erythropoiesis contributes significantly to 
anemia and overall disease morbidity.  
7.2. 
Summary of Nonclinical Studies 
A brief summary of key findings from pharmacology and toxicology studies is provided below.  
A comprehensive review of ACE-536, as well as details regarding the information summarized 
below, is provided in the Investigator’s Brochure (IB).  The most recent version of the ACE-536 
IB should be reviewed prior to initiating the study. 
7.2.1. 
Pharmacology Studies 
In vitro and in vivo pharmacology studies have been conducted with ACE-536 or its murine 
orthologue, RAP-536.  The use of RAP-536 allows longer term pharmacology studies in rodents 
without the confounding influence of immune reactivity. 
A total of 26 potential ligands within the TGF-β superfamily were assessed for their in vitro 
binding specificity and affinity to ACE-536 by surface plasmon resonance (Biacore).  Among the 
ligands examined, GDF8, GDF11, BMP6, and activin B bound to ACE-536 with the highest 
affinities at 37°C (KD values in the sub-nanomolar range).  Further studies using a cell line 
reporter gene assay to assess signaling through the activin receptor demonstrate that signaling by 
GDF11 and GDF8 was inhibited by ACE-536. 


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During the course of early nonclinical pharmacology studies, an observation of a rapid, robust, 
and sustained increase in erythropoiesis in animals treated with ACE-536 was made.  The effect 
of ACE-536 on bone marrow erythroid progenitor cells in mice was assessed by FACS analysis.  
Results demonstrate that the increase in RBC results as a consequence of a decrease of 
basophilic erythroblasts and a coordinate increase in subsequent, later stage polychromatic and 
orthochromatic erythroblasts as well as nucleated reticulocytes in both the bone marrow and 
spleen.  Separate studies have confirmed that the increase in RBC occurs without affecting the 
populations of erythropoietin (EPO) responsive cells (BFU-E and CFU-E).   
Fundamental to this application, a series of studies have been conducted in Hbbmajor-/- mice, a 
model of β-thalassemia in humans.  These mice carry a deletion mutation in the β-globin gene, 
resulting in hematologic abnormalities very similar to those seen in human β-thalassemia 
intermedia, including severe anemia, ineffective erythropoiesis, splenomegaly and iron 
overload.8  Results of studies in Hbbmajor-/- mice, described in more detail below, demonstrate that 
treatment with ACE-536 led to significant improvements in hematologic parameters, decreased 
hemolysis and reductions in spleen weight.  Further, ACE-536 was shown to influence erythroid 
differentiation in the bone marrow and spleen of Hbbmajor-/- mice to increase the number of 
late-stage precursor cell populations. 
RAP-536 was evaluated for its ability to improve ineffective erythropoiesis in thalassemic mice.  
Hbbmajor-/- mice, or their wild-type (Wt) counterparts, were treated with RAP-536 (1 mg/kg SC) 
or Tris-buffered saline (TBS) control, twice weekly for 8 weeks.  Mice were 3 months of age at 
the initiation of the study and clearly exhibited features of β-thalassemia.  At the end of the 
8-week treatment period, assessment of hematologic parameters showed that RAP-536 treatment 
resulted in significant increases in red cell count, hemoglobin levels (Figure 1) and hematocrit, 
normalized red cell distribution width, and reduced red cell hemolysis, as measured by serum 
total bilirubin. 
Importantly, the mechanism of RAP-536 action in Hbbmajor-/- mice was shown to be related to the 
accelerated maturation of erythroid precursor cell populations, specifically, increases in the 
orthochromatic erythroblasts and reticulocytes in the bone marrow and spleen, with concomitant 
reductions in the earlier-stage basophilic erythroblast population. 
Concurrent with improvements in the hematology profile and in bone marrow/spleen cytology, 
Hbbmajor-/- mice treated with RAP-536 showed significant reductions in absolute spleen weight 
and as a percentage of body weight (data not shown).  Finally, an unexpected finding from this 
study was an increase in bone mineral density, as measured by whole body DEXA scans, of 
Hbbmajor-/- mice treated with RAP-536.  The mechanism for increased bone density is presently 
unclear, but could be related to normalization of bone metabolism associated with decreased 
anemia and concomitantly reduced marrow expansion cohort in RAP-536 treated animals, or 
possibly due to a direct effect of RAP-536 on bone turnover. 
 




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7.3. 
Summary of Clinical Experience 
The safety, tolerability, pharmacokinetics, and pharmacodynamic effects of ACE-536 was 
evaluated in healthy postmenopausal women in a phase 1, randomized, double-blind, placebo 
controlled, multiple ascending dose study (Study A536-02).  The primary objective of the study 
is to evaluate the safety and tolerability of ACE-536 in this population.  Secondary objectives of 
the study are to examine the pharmacokinetic parameters and pharmacodynamic effects of 
ACE-536.  Preliminary data from Study A536-02 as of 05 July 2012 are summarized below. 
A total of 32 subjects were enrolled.  The study consisted of 4 cohorts (0.0625 mg/kg x 2 doses, 
0.125 mg/kg x 1 dose, 0.125 mg/kg x 2 doses, and 0.25 mg/kg x 2 doses).  Subjects in each 
cohort were randomized and receive either ACE-536 or placebo (6 active and 2 placebo subjects 
per cohort).  Doses were administered SC on Day 1 and Day 15.  One subject in the third cohort 
and 5 subjects in the fourth cohort received only one dose of ACE-536. 
The mean (SD) age for subjects in this study was 59.4 (5.8) years.  Mean baseline hemoglobin 
level was 13.2 (0.6) g/dL.  Preliminary analysis of PK data demonstrated that maximum 
ACE-536 concentration (Cmax) and area under the curve (AUC0-14d) after the first dose were 
generally dose-proportional from 0.0625 to 0.25 mg/kg.  The mean time to maximum 
concentration (Tmax) ranged from 7.7 to 11.7 days and the mean elimination half-life (t½) ranged 
from 15.5 to 18.5 days.  Pharmacokinetic results support SC dosing of ACE-536 every 3 weeks.    
ACE-536 was generally safe and well-tolerated in this study of healthy postmenopausal female 
subjects.  No serious or severe adverse events were reported.  The majority of AEs were 
considered mild in severity.  Adverse events that were considered probably or possibly related to 
study drug included injection site hemorrhage (3 subjects on active drug), injection site macule 
(2 subjects), and dry skin, hyperesthesia, muscle spasms, myalgia, generalized pruritis, and 
papular rash (1 subject each). 
Consistent with results from the nonclinical pharmacology studies, ACE-536 administration 
resulted in a mean maximum increase in hemoglobin (at any timepoint during the study) of 
1.3 g/dL in the 0.25 mg/kg group.  The maximum hemoglobin increase from baseline at any 
timepoint for patients in the 0.25 mg/kg group ranged from 0.6 to 2.0 g/dL.  The mean 
hemoglobin increase from baseline in that treatment group was at least 0.6 g/dL from Day 8 
through Day 57, compared with a mean hemoglobin decrease from baseline in the placebo group 
of up to 0.6 g/dL through Day 43 (Figure 2). 


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Figure 2: 
Mean (±SD) Change from Baseline of Plasma Hemoglobin (g/dL) in Healthy 
Volunteers Treated with One or Two Doses of ACE-536 
 
The preliminary results from this phase 1 study suggest a positive effect of 0.25 mg/kg ACE-536 
on increasing hemoglobin in healthy subjects.  Doses up to 0.25 mg/kg were generally safe and 
well-tolerated. 
7.4. 
Potential Risks of Human Use 
Increases in hematologic parameters (RBC, hemoglobin, hematocrit, reticulocytes) are expected 
pharmacologic effects of ACE-536 treatment.  Increases in systolic and diastolic blood pressures 
may occur in concert with increases in hemoglobin values.  Excessive or rapid increases in 
hemoglobin or blood pressure may occur and will be monitored.  Dose modifications rules for 
individual subjects and dose escalation/reduction rules will be utilized to minimize risks 
associated with increased RBC parameters. 
Adverse events observed in the Phase 1 study in healthy volunteers that were considered 
probably or possibly related to study drug included injection site hemorrhage, injection site 
macule, dry skin, hyperesthesia, muscle spasms, myalgia, generalized pruritis, and papular rash. 
Membranoproliferative glomerulonephritis was observed in both rats and monkeys, likely 
secondary to immune complex deposition.  These findings were associated with increases in 
serum BUN and creatinine.  The presence of immune complexes in renal tissue from monkeys 
treated with ACE-536 was demonstrated by immunohistochemistry.  
The growth of heart and lungs was slightly reduced in rats exposed to ACE-536 as compared 
with control animals; these decreased heart and lung weights were reversible and there was no 


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corresponding histology finding.  These findings were not replicated in the monkey toxicology 
studies; the potential for these adverse effects in humans is unknown.  
Based on rat toxicology studies, treatment with ACE-536 may affect the adrenal gland.  These 
findings were specific to the rat as they were not replicated in the monkey toxicology studies.  
As with all biologics, there is the potential for anti-drug antibodies that can be associated with 
increased drug clearance and hypersensitivity reactions.  Anti-drug antibody formation against 
ACE-536 as well as human ActRIIB protein will be monitored in the initial clinical studies. 
No reproductive toxicity studies have been conducted with ACE-536 and therefore ACE-536 
should not be administered to pregnant women.  Male and female subjects of childbearing 
potential participating in studies of ACE-536 must be willing to abstain from sexual intercourse 
or use adequate contraception during the treatment and for 12 weeks following the last dose of 
ACE-536.  The effects of ACE-536 on the developing fetus are unknown.  It is not known if 
ACE-536 is secreted in milk; therefore, ACE-536 should not be administered to nursing mothers.  
It is unknown if humans will experience any of the effects of ACE-536 that were noted in the rat 
and monkey toxicology studies.  Safety effects will be monitored closely through adverse event 
reporting, clinical laboratory tests, vital signs, and physical examinations. 
A comprehensive review of ACE-536, as well as details regarding the information summarized 
above, is provided in the Investigator’s Brochure (IB).  The ACE-536 IB should be reviewed 
prior to initiating the study. 


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8. 
TRIAL OBJECTIVES 
8.1. 
Primary Objective  
 To evaluate the proportion of β-thalassemia patients who have an erythroid response, 
defined as:  1) a hemoglobin increase of ≥ 1.5 g/dL from baseline for ≥ 14 days (in 
the absence of red blood cell [RBC] transfusions) in non-transfusion dependent 
patients, or 2) ≥ 20% reduction in RBC transfusion burden compared to pretreatment 
in transfusion dependent patients  
8.2. 
Secondary Objectives 
 To evaluate safety and tolerability of ACE-536 
 To evaluate the proportion of transfusion dependent patients who have ≥ 50% 
reduction of RBC transfusion burden compared to pretreatment  
 To evaluate the time to erythroid response and duration of erythroid response 
 To evaluate the change in hemoglobin level in non-transfusion dependent patients 
 To evaluate the change in pre-transfusion hemoglobin levels in transfusion dependent 
patients 
 To evaluate changes in biomarkers of erythropoiesis, hemolysis, iron metabolism, and 
bone metabolism  
 To examine the pharmacokinetic (PK) profile of ACE-536 in patients with 
β-thalassemia  
8.3. 
Exploratory Objective 
 To evaluate biomarkers related to the TGF- superfamily 
 To evaluate self-reported quality of life using tools including but not limited to the 
FACT-An and SF-36 questionnaires (expansion cohort only) 
 To evaluate change in spleen size by MRI 
 To evaluate change in bone mineral density (BMD) by DXA 
 To evaluate change in extramedullary hematopoiesis (EMH) mass size by MRI 
 To evaluate change in leg ulcer size 
 To evaluate change in the 6-minute walk test (6MWT) distance in NTD patients 


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9. 
OVERALL STUDY DESIGN AND PLAN: DESCRIPTION 
This is a phase 2, open-label, ascending dose study to evaluate the effects of ACE-536 in patients 
with β-thalassemia.  
9.1. 
Study Design  
Patients who meet the study eligibility criteria will be enrolled within 28 days of screening.  
Patients in all cohorts will receive ACE-536, administered subcutaneously (SC), every 3 weeks 
for up to 5 cycles.  ACE-536 cannot be administered after study day 85 (± 2 days).  Dose 
delay(s) and dose reduction(s) may be required for individual patients as outlined in the Patient 
Dose Modification Rules (Section 10.8.1).   
Each dose escalation cohort will consist of up to 6 patients.  The dose level of ACE-536 for the 
first cohort will be 0.2 mg/kg and the dose level(s) for subsequent cohort(s) will follow a 
modified Fibonacci dose escalation scheme (i.e., maximum dose of 0.4 mg/kg for cohort 2, 
0.6 mg/kg for cohort 3, 0.8 mg/kg for cohort 4, 1.0 mg/kg for cohort 5, etc.).  Once a minimum 
of 3 patients in a cohort have completed Study Day 29, a Safety Review Team (SRT) will review 
preliminary safety and hematologic response data and make recommendations to the Sponsor 
regarding whether or not to enroll an additional cohort at a higher or lower dose, complete 
enrollment of 6 patients at the current dose level, or proceed to the expansion cohort.   
The expansion cohort (n=30) will be treated with ACE-536 at a starting dose level not to exceed 
the maximum dose level for the previous cohorts.  The expansion cohort will include a minimum 
of 12 patients who are non-transfusion dependent and a minimum of 12 patients who are 
transfusion dependent (including a minimum of 6 thalassemia major patients with onset of 
regular transfusions before age 4 years, and a minimum of 6 patients with onset of regular 
transfusions onset after age 4 years).  For subsequent cycles in the expansion cohort, a patient’s 
dose level may be titrated from the previous dose administered for that patient.  Patients in the 
expansion cohort only may have their dose level titrated up or down as required per protocol to 
meet dose titration and modification rules (see Section 10.8).  The maximum dose level given to 
a patient will not exceed the maximum dose level evaluated in the dose escalation cohorts.  
Patients will be treated with up to 5 doses of ACE-536.  
Each TD patient in the expansion cohort will have a defined “pre-transfusion hemoglobin 
threshold” which will be calculated based on transfusion history and will be used for determining 
when to transfuse during the study.  The baseline pre-transfusion hemoglobin threshold will be 
the mean of all documented pre-transfusion hemoglobin values during the 12 weeks prior to 
C1D1.  During treatment, if the pre-transfusion hemoglobin value is increased by ≥ 1 g/dL 
compared to the baseline pre-transfusion hemoglobin threshold for that patient, transfusion 
should be delayed by a minimum of 7 days and/or the number of units transfused should be 
reduced by 1 or more RBC units.  Patients may be transfused at the investigator’s discretion for 
symptoms related to anemia or other requirements (e.g., infection). 
 


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Cohort
a 
ACE-536 Dose Level
b 
(mg/kg) 
Number of 
Patients 
1 
0.2 
3-6 
2 
0.4 
3-6 
3 
0.6 
3-6 
4 
0.8 
3-6 
5 
1.0 
3-6 
6 
1.25 
3-6 
7 
1.5 
3-6 
Expansion 
TBD 
30
c 
Planned Total (All Cohorts):  
 
Approximately 51-72 
a  Cohort escalation is based on SRT review and recommendation to enroll additional cohorts and/or the expansion cohort. 
b The ACE-536 dose level for cohort 1 is 0.2 mg/kg.  The dose level indicated in the table for all subsequent cohorts is the 
maximum dose level that can be recommended by the SRT for escalation per the modified Fibonacci dose escalation scheme. 
c The expansion cohort will consist of 30 patients, including a minimum of 12 patients who are non-transfusion dependent and a 
minimum of 12 patients who are transfusion dependent (including a minimum of 6 thalassemia major patients with onset of 
regular transfusions before age 4 years, and a minimum of 6 patients with onset of regular transfusions after age 4 years).   
9.2. 
Discussion of Study Design 
The primary efficacy endpoint, evaluating sustained increase in hemoglobin during treatment 
with ACE-536, addresses the primary deficiency in -thalassemia, namely profound anemia 
particularly marked in thalassemia major, but clearly present also in thalassemia intermedia and 
largely driving the clinical picture.  A therapy capable of maintaining or increasing hemoglobin 
levels with chronic treatment can reverse the symptoms associated with anemia, reduce 
transfusion requirement, decrease iron overload, and decrease the need for iron chelation and the 
associated adverse effects of chelation treatment.  ACE-536 treatment may prove effective in 
promoting erythroid maturation and reducing ineffective erythropoiesis.  It may have additional 
salutary effects on iron metabolism and bone defects.  It is unknown if ACE-536 will also affect 
the globin chain imbalance characteristic of the disease.  Thus, the secondary endpoints were 
selected to evaluate the impact of ACE-536 on hematology, erythropoiesis, iron metabolism, and 
bone metabolism parameters. 
The initial dose level of 0.2 mg/kg is less than the maximum dose administered in the phase 1 
study A536-02 of 0.25 mg/kg, which was safe and well-tolerated, and produced a hemoglobin 
response in healthy volunteers.  The safety measurements in this study are standard for studies 
with investigational medications.  An open-label study, conducted in a limited number of 
patients, with protocol-specified definitions of dose-limiting toxicity, and with protocol-specified 
procedures for dose escalation and reduction is considered appropriate for a proof-of-concept 
study in this target patient population.  Dose escalation in sequential cohorts is designed to 
minimize the risk of adverse events as well as excessive erythroid response and hypertension that 
may result from a robust response to high drug levels.  Individualization of dose level in the 
expansion cohort, using dose titration rules based on hemoglobin response and dose reduction 


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rules based on safety information will maximize the proportion of patients having an erythroid 
response while maintaining safety. 
9.3. 
Selection of Study Population 
9.3.1. 
Inclusion Criteria 
Eligible patients must meet all of the following criteria: 
1. Men or women  18 years of age. 
2. For the dose escalation phase of the study:  documented diagnosis of β-thalassemia 
intermedia (transfusion dependent patients must not have begun regular transfusions at 
age < 4.0 years)  
For the expansion cohort:  documented diagnosis of β-thalassemia (including 
β-thalassemia major or β-thalassemia intermedia). 
3. Prior splenectomy or spleen size < 18 cm in the longest diameter by abdominal 
ultrasound (dose escalation cohorts only).   
4. Anemia, defined as: 
a. Mean hemoglobin concentration < 10.0 g/dL of 2 measurements (one performed 
within one day prior to Cycle 1 Day 1 and the other performed during the 
screening period [Day -28 to Day -1]) in non-transfusion dependent patients, 
defined as having received < 4 units of RBCs within 8 weeks prior to Cycle 1 Day 
1, OR 
b. Transfusion dependent, defined as requiring ≥ 4 units of RBCs every 8 weeks 
(confirmed over 6 months prior to Cycle 1 Day 1).  
5. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) < 3 x upper limit of 
normal (ULN). 
6. Serum creatinine ≤ 1.5 x ULN. 
7. Females of child bearing potential (defined as sexually mature women who have not 
undergone hysterectomy or bilateral oophorectomy, or are not naturally postmenopausal 
≥ 24 consecutive months) must have negative urine or blood pregnancy test prior to 
enrollment and use adequate birth control methods (abstinence, oral contraceptives, 
barrier method with spermicide, or surgical sterilization) during study participation and 
for 12 weeks following the last dose of ACE-536.  Males must agree to use a latex 
condom during any sexual contact with females of child-bearing potential during study 
participation and for 12 weeks following the last dose of ACE-536, even if he has 
undergone a successful vasectomy.  Patients must be counseled concerning measures to 
be used to prevent pregnancy and potential toxicities prior to the first dose of ACE-536. 
8. Patients are able to adhere to the study visit schedule, understand and comply with all 
protocol requirements. 
9. Understand and able to provide written informed consent. 


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9.3.2. 
Exclusion Criteria 
Eligible patients must not meet any of the following criteria: 
1. Any clinically significant pulmonary (including pulmonary hypertension), 
cardiovascular, endocrine, neurologic, hepatic, gastrointestinal, infectious, 
immunological (including clinically significant allo- or auto-immunization), or 
genitourinary disease considered by the investigator as not adequately controlled prior to 
Cycle 1 Day 1.  
2. Folate deficiency. 
3. Symptomatic splenomegaly. 
4. Known positive for human immunodeficiency virus (HIV), active infectious hepatitis B 
(HBV), or active infectious hepatitis C (HCV). 
5. Known history of thromboembolic events ≥ grade 3 according to the National Cancer 
Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) v.4.0 (current 
active minor version). 
6. Ejection fraction < 50% by echocardiogram, MUGA or cardiac MRI. 
7. Uncontrolled hypertension defined as systolic blood pressure (BP) ≥ 150 mm Hg or 
diastolic BP ≥ 95 mm Hg. 
8. Heart failure class 3 or higher (New York Heart Association, NYHA, Appendix 1). 
9. QTc > 450 msec on screening ECG. 
10. Platelet count < 100 x109/L or > 1,000 x109/L. 
11. Proteinuria ≥ Grade 2. 
12. Any active infection requiring parenteral antibiotic therapy within 28 days prior to 
Cycle 1 Day 1 or oral antibiotics within 14 days of Cycle 1 Day 1. 
13. Treatment with another investigational drug or device, or approved therapy for 
investigational use ≤ 28 days prior to Cycle 1 Day 1, or if the half-life of the previous 
investigational product is known, within 5 times the half-life prior to Cycle 1 Day 1, 
whichever is longer.  
14. Transfusion event within 7 days prior to Cycle 1 Day 1. 
15. Patients receiving or planning to receive hydroxyurea treatment.  Patients must not have 
had hydroxyurea within 90 days of Cycle 1 Day 1.    
16. Splenectomy within 56 days prior to Cycle 1 Day 1. 
17. Major surgery (except splenectomy) within 28 days prior to Cycle 1 Day 1.  Patients must 
have completely recovered from any previous surgery prior to Cycle 1 Day 1. 
18. Iron chelation therapy initiated within 56 days prior to Cycle 1 Day 1. 
19. Cytotoxic agents, systemic corticosteroids, immunosuppressants, or anticoagulant therapy 
such as warfarin or heparin within 28 days prior to Cycle 1 Day 1 (prophylactic aspirin 
up to 100 mg/day is permitted). 


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20. Pregnant or lactating females.  
21. History of severe allergic or anaphylactic reactions or hypersensitivity to recombinant 
proteins or excipients in the investigational drug. 
22. Prior treatment with sotatercept (ACE-011) or ACE-536. 
9.4. 
Patient Treatment Discontinuation and Withdrawal Criteria 
Patients will be informed that they have the right to withdraw from the study at any time for any 
reason without prejudice to their medical care.   
A patient may be discontinued from treatment for any of the following reasons:  
 Patient’s request 
 Patient’s unwillingness or inability to comply with the protocol 
 Pregnancy 
 Use of prohibited medication (e.g., hydroxyurea) 
 Medical reason or adverse event, at the discretion of the investigator and/or the 
medical monitor 
 Hypersensitivity reaction to study drug 
 At the discretion of the sponsor (e.g., termination of the study or a dose level) 
A patient may be withdrawn from the study for any of the following reasons: 
 Patient’s request 
 Patient’s unwillingness or inability to comply with the protocol 
 Death 
 Loss to follow-up 
 At the discretion of the sponsor (e.g., termination of the study) 
The reasons for study withdrawal and treatment discontinuation must be recorded in the patient’s 
CRF.  The investigator must notify the sponsor, the medical monitor and the contract research 
organization (CRO) immediately when a patient has been discontinued/withdrawn due to an AE.  
Patients who discontinue treatment early should complete the end of treatment (EOT) follow-up 
visit at the time of discontinuation and then complete the end of study (EOS) follow-up visit 
approximately 28 days later.   
The investigator must notify the sponsor and the CRO when a patient has been 
discontinued/withdrawn for reasons unrelated to the study or study drug (e.g., withdrawn 
consent, lost to follow up).  


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10. 
TREATMENT OF PATIENTS 
10.1. 
Selection and Timing of Dose for Each Patient 
Once a patient is enrolled, the appropriate dose of ACE-536 will be administered as a 
subcutaneous injection on Cycle 1 Day 1.  Subsequent doses will be administered every 3 weeks 
on Day 1 of the cycle.  Study day 85 (± 2 days) is the last possible study day that ACE-536 can 
be administered, regardless of the cycle.  Dose reductions may be required for individual patients 
as outlined in the Patient Dose Modifications Rules (Section 10.8.1).  If a dose delay is required, 
the patient will return weekly for assessment of hematology results and adverse events until the 
patient is eligible to receive the next dose of ACE-536.  Patients will be asked to return to the 
clinic for two follow-up visits, occurring approximately 28 days and 56 days after their last dose 
of ACE-536.  If a patient has a positive anti-drug antibody (ADA) result at the last visit, the 
patient may be asked to return for additional ADA testing every three months, until a negative 
result is obtained or the result is considered to be stabilized. 
10.2. 
Concomitant Medications 
During screening and throughout the study patients may take stable doses of medications for 
chronic conditions that are not specifically excluded by the protocol (see Section 9.3.1 and 
Section 9.3.2).  If a patient requires treatment with any new medications that are specifically 
excluded by the eligibility criteria, the patient will be discontinued from the study and should 
complete the end of treatment visit and enter the follow-up period of the study.  The investigator 
should consult the medical monitor regarding any questions about whether a new medication or 
dosage of existing medication would require the patient to discontinue from the study.  
Concomitant medications will be collected beginning at study screening and will include all 
medications taken within 28 days prior to Cycle 1 Day 1.  
10.2.1. 
Other Treatments for β-Thalassemia  
If treatment with hydroxyurea is required during the ACE-536 treatment period, the patient 
should be discontinued from treatment with study drug and complete the end of treatment visit 
and enter the follow-up period.  Iron chelation therapy is allowed if initiated at least 56 days 
prior to C1D1. 
10.2.2. 
RBC Transfusions 
Concurrent treatment for anemia with blood transfusions is allowed at the discretion of the 
investigator, for low hemoglobin levels, symptoms associated with anemia (e.g., hemodynamic 
or pulmonary compromise requiring treatment) or a comorbidity. 
For any RBC transfusions received during the study, collect hemoglobin values just prior to 
transfusion. 
Each TD patient in the expansion cohort will have a defined “pre-transfusion hemoglobin 
threshold” which will be calculated based on transfusion history and will be used for determining 
when to transfuse during the study.  The baseline pre-transfusion hemoglobin threshold will be 
the mean of all documented pre-transfusion hemoglobin values during the 12 weeks prior to 


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C1D1.  During treatment, if the pre-transfusion hemoglobin value is increased by ≥ 1 g/dL 
compared to the baseline pre-transfusion hemoglobin threshold for that patient, transfusion 
should be delayed by a minimum of 7 days and/or the number of units transfused should be 
reduced by 1 or more RBC units.  Patients may be transfused at the investigator’s discretion for 
symptoms related to anemia or other requirements (e.g., infection). 
For the first dose of ACE-536, dosing should occur after a minimum of 7 days post-transfusion; 
subsequent doses should not be given within 24 hours of transfusion or planned transfusion.   
10.3. 
Treatment Compliance 
ACE-536 will be administered as a SC injection at the clinical site by the study staff and will be 
documented in the study record.  Monitoring for patient compliance with the treatment regimen 
is therefore unnecessary.  
10.4. 
Randomization  
This is an open-label study that does not require randomization. 
10.5. 
Treatments Administered 
Patients will continue to receive the same dose level of study drug as they were assigned at study 
entry unless a dose modification is required (see Section 10.8).   
10.6. 
Dose-Limiting Toxicity Definition  
A Dose-Limiting Toxicity (DLT), using the current active minor version of the National Cancer 
Institute Common Toxicity Criteria for Adverse Events version 4.0 (NCI-CTCAE v4.0), is 
defined as any of the following toxicities, at any dose level occurring within 28 days of the first 
administered dose: 
 Treatment related serious adverse event (SAE) of Grade ≥ 3  
 Treatment related non-hematologic adverse event (AE) of Grade ≥ 3 
 Treatment related hematologic AE of Grade ≥ 4. 
10.7. 
Safety Evaluation and Dose Escalation 
Safety will be evaluated by the Safety Review Team (SRT), which is comprised at minimum of 
the coordinating investigator, medical monitor, and an independent hematologist.  The role of the 
SRT will be described in greater detail in the SRT Charter.  The SRT will review safety data 
when a minimum of 3 patients in a cohort have completed Study Day 29, including dose-limiting 
toxicities (DLTs), adverse events (AEs), serious adverse events (SAEs), laboratory results 
(including hematology and chemistry), vital signs, and erythroid response data.  The SRT may 
make one or more of the following recommendations to the sponsor: 
 Enroll 3-6 patients in an additional cohort at a higher or lower dose level, not to 
exceed modified Fibonacci dose escalation scheme (see Section 9.1); enrollment in 
current cohort may be closed after a minimum of 3 patients. 


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 Proceed to the expansion cohort, with a recommended starting dose level for TD and 
NTD patients, not to exceed the maximum dose level evaluated in the dose escalation 
cohorts. 
 Postpone a decision pending collection of additional data from the current cohort. 
 Recommend that no further cohorts should be enrolled in the study. 
SRT recommendations to escalate or reduce the dose level of ACE-536 in a subsequent cohort, 
or complete enrollment of 6 patients in the current cohort, will be based in part upon the 
following criteria: 
 If a DLT occurs in ≤ 1 patient in a cohort within 28 days following the initial dose of 
ACE-536, dose escalation may occur; 
 If a DLT occurs in ≥ 2 patients in a cohort within 28 days of the initial dose, the dose 
level for the next cohort (if any) should not be escalated; a lower dose may be 
recommended. 
 If a hemoglobin increase of ≥ 2.0 g/dL (confirmed within 48 to 72 hours and not 
attributable to RBC transfusion) occurs in ≥ 2 patients in a cohort within 28 days of 
the initial dose, the dose level for the next cohort (if any) should not be escalated; a 
lower dose may be recommended. 
10.8. 
Patient Dose Modification and Titration 
Patients in the dose escalation cohorts have a pre-determined dose level based on cohort number 
(see Section 9.1).  Dose level titration is not allowed for patients in the dose escalation cohorts, 
but dose delay or reduction may be required according to Patient Dose Modification Rules 
(Section 10.8.1).  Examples of dose level reductions for patients in dose escalation cohorts are 
included in Table 2 below.  Patients who require more than 2 dose reductions due to an AE 
should be discontinued from treatment and complete the end of treatment and end of study visits.   
All patients in the expansion cohort will be initially treated with ACE-536 at an assigned starting 
dose level (to be determined) which has been deemed to be safe, well tolerated, and at least 
minimally effective by the SRT.  Examples of possible expansion cohort starting dose levels and 
associated modifications (reductions and titrations) are in Table 2.  Rules for individual patient 
titrations are described in Section 10.8.2 for NTD patients and Section 10.8.3 for TD patients.  
 
 


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Table 2: 
Starting Dose Levels, Dose Level Reductions (All Cohorts), and Dose Level 
Titrations (Expansion Cohort Only) 
2nd Dose 
Reduction 
1st Dose 
Reduction 
Starting 
Dose Level 
1st Dose 
Titration 
2nd Dose 
Titration 
0.2 mg/kg 
0.4 mg/kg 
0.6 mg/kg 
0.8 mg/kg 
1.0 mg/kg 
0.4 mg/kg 
0.6 mg/kg 
0.8 mg/kg 
1.0 mg/kg 
1.25 mg/kg 
0.6 mg/kg 
0.8 mg/kg 
1.0 mg/kg 
1.25 mg/kg 
1.5 mg/kg 
0.8 mg/kg 
1.0 mg/kg 
1.25 mg/kg 
1.5 mg/kg 
N/A 
1.0 mg/kg 
1.25 mg/kg 
1.5 mg/kg 
N/A 
N/A 
 Dose titration to higher dose levels is only applicable to patients in the expansion 
cohort. 
 Expansion cohort starting dose level for TD and NTD patients to be determined by 
the sponsor in conjunction with the SRT (range: 0.2-1.5 mg/kg). 
 Dose titration will not exceed the maximum dose level tested in the dose escalation 
cohorts and determined to be safe and well tolerated by the SRT. 
 Patients may increase and decrease dose level per protocol as needed, however 
patients who require more than 2 dose reductions due to an AE should be 
discontinued from treatment and complete the end of treatment and end of study 
visits. 
10.8.1. 
Patient Dose Modification Rules 
The following dose modification rules include both pharmacodynamic and safety parameters 
which may require a dose delay and possibly a dose reduction.  These rules should be assessed 
prior to dosing.  If a dose delay is required the patient should have weekly visits to assess 
hematology results and adverse events until the patient is eligible to receive the next dose of 
ACE-536.   
Observation on Dosing Day 
Action 
ACE-536 Dose Modification
a 
Hemoglobin ≥ 13 g/dL 
Hold dose and monitor 
patient weekly until 
hemoglobin < 12 g/dL 
Resume dosing once hemoglobin 
< 12 g/dL.   
Hemoglobin increase ≥ 2 g/dL from 
the previous dosing day (not 
attributable to RBC transfusion) 
Continue dosing 
Reduce dose by 1 dose levelb 
Non-hematologic adverse event 
≥ Grade 3, or hematologic adverse 
event ≥ Grade 4, at least possibly 
related to study drug 
Hold dose and monitor 
patient weekly until 
resolution of AE to 
≤ Grade 1 or baseline 
Resume dosing upon resolution of the AE 
to ≤ Grade 1 or baseline and reduce dose 
level by 1 dose levelb 
a Last possible treatment day is Study Day 85 (± 2 days).  
b  All subsequent doses will remain at the new dose level or can be reduced further if needed.  Patients who require more than 2 
dose reductions due to an AE should be discontinued from treatment and complete the end of treatment and end of study visits. 


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10.8.2. 
Individual Dose Titration Rules for NTD Patients in the Expansion Cohort 
For the expansion cohort, the starting dose to be administered on Cycle 1 Day 1 for NTD patients 
will be determined by the sponsor in conjunction with the SRT (see Table 2).  For subsequent 
cycles, dose level may be titrated individually for each patient.  For all NTD patients the dose 
level will be assessed for titration at C3D1, C4D1, and C5D1.  The dose level should be titrated 
individually for each patient not to exceed the maximum dose level evaluated as follows: 
 If hemoglobin increase from baseline is < 1.5 g/dL throughout the previous 2 cycles at 
the same dose level, the dose level will be increased by 1 dose level (unless dose 
modification is required). 
 If hemoglobin increase from baseline is ≥ 1.5 g/dL but not sustained for at least 2 
consecutive measurements during the previous 2 cycles at the same dose level, the dose 
level will be increased by 1 dose level (unless dose modification is required). 
 If hemoglobin increase from baseline is ≥ 1.5 g/dL for at least 2 consecutive 
measurements during the previous 2 cycles at the same dose level, the dose level will be 
unchanged (unless dose modification is required). 
Baseline hemoglobin to assess titration will be the average of 2 or more measurements 
performed during the screening period.  Hemoglobin measurements within 2 weeks following 
RBC transfusion will be excluded from the both the calculated mean baseline hemoglobin and on 
study values used for evaluating dose titration for a patient. 
10.8.3. 
Individual Dose Titration Rules for TD Patients in the Expansion Cohort 
For the expansion cohort, the starting dose to be administered on Cycle 1 Day 1 for TD patients 
will be determined by the sponsor in conjunction with the SRT (see Table 2Table 2).  For 
subsequent cycles, the dose level may be titrated individually for each patient.  For all TD 
patients the dose level will be assessed for titration at C3D1, C4D1, and C5D1.  The dose level 
should be titrated individually for each patient not to exceed the maximum dose level evaluated 
as follows: 
 If patient is transfused with ≥ 3 units during the previous 2 cycles at the same dose level, 
the dose level will be increased by 1 dose level (unless a dose modification is required) 
A dose delay and/or dose reduction may be required for individual patients as outlined in the 
Patient Dose Modification Rules (Section 10.8.1).  The SRT will meet periodically to monitor 
overall safety and erythroid response data for the expansion cohort. 
10.8.4. 
Other Considerations for Dose Modification, Delay or Discontinuation 
For individual patients judged by the investigator to be at an unacceptable risk, but who do not 
meet the protocol-defined conditions for a dose modification, the investigator should consult 
with the medical monitor to decide whether to continue dosing at the same dose level, reduce the 
dose level, or discontinue the patient’s treatment with ACE-536. 


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11. 
STUDY PROCEDURES  
Please refer to Section 2 for the schedule of procedures required for each visit. 
11.1. 
Written Informed Consent 
Patients will be required to sign an IEC approved ICF prior to any study related procedures, 
including screening evaluations. 
11.2. 
Clinical Laboratory Tests 
The following laboratory assessments will be performed at the clinical site’s local laboratory 
according to the laboratory collection recommendations.  The sponsor may request additional 
safety tests be performed based on ongoing data review during the study.  
 Hematology: RBC, WBC with differential, hemoglobin, hematocrit, haptoglobin 
(optional), reticulocyte count, platelet count, MCV, MCH, MCHC, and RDW.   
 Serum chemistry: Sodium, potassium, chloride, carbon dioxide/bicarbonate 
(optional), AST, ALT, lactate dehydrogenase (LDH), total bilirubin, indirect 
bilirubin, alkaline phosphatase, blood urea nitrogen (BUN)/urea, creatinine, GGT, 
calcium, phosphorus, glucose, amylase, lipase, total protein, albumin, and uric acid. 
 Urinalysis by dipstick analysis: pH, specific gravity, protein, glucose, ketones, blood, 
leukocyte esterase, and nitrite, with microscopic examination if indicated. 
 Pregnancy test (urine or serum) and menstrual history for women of child bearing 
potential. 
 Screening visit only: serum folate, B12  
The following laboratory assessments will be performed at the central laboratory according to 
the laboratory collection recommendations.  The sponsor may request additional safety tests be 
performed based on ongoing data review during the study.  
 Iron studies: Serum iron, TIBC, transferrin, soluble transferrin receptor, ferritin, 
NTBI, and hepcidin. 
 nRBCs 
 Erythropoietin levels 
 Peripheral blood smear 
 Hemoglobin electrophoresis, globin chain mRNA 
 Urine microalbumin and creatinine 
 Bone Biomarkers:  serum BSAP and CTX  
11.3. 
Other Safety Assessments   
 Physical examination (including optional evaluation of gonadal size in males) 


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 Vital signs: weight, heart rate, systolic and diastolic blood pressure, respiration rate, 
and temperature (measured in degrees Celsius).  Height is measured only at 
screening.  If at any visit the systolic blood pressure is ≥ 150 mmHg, the diastolic 
blood pressure is ≥ 95 mmHg, and/or the absolute increase in either measure from 
baseline is ≥ 20 mmHg, perform one repeat blood pressure assessment after a 
minimum of 15 minutes. 
 12-lead ECG  
 Anti-drug antibody testing   
 Abdominal ultrasound should be performed as needed for patients with clinical signs 
of a change in spleen size or abnormality in the dose escalation cohorts only. 
11.4. 
Pharmacokinetic and Pharmacodynamic Assessments 
11.4.1. 
Pharmacokinetic Assessments 
Pharmacokinetic assessment of ACE-536 concentrations will be performed periodically as 
outlined in Section 2.  Blood samples should be drawn and processed on-site for serum collection 
at the time points specified.  Additional details regarding PK collection and processing can be 
found in the Study Reference Guide.   
11.4.2. 
Pharmacodynamic Evaluations 
Pharmacodynamic assessments including hematologic laboratory assessments and transfusion 
information will be used to determine erythroid response to ACE-536. 
An MRI will be performed at selected sites to evaluate liver iron content (all cohorts), EMH 
mass size (expansion cohort only), and spleen size (expansion cohort only). 
A DXA will be performed at selected sites to evaluate total body, lumbar spine and total hip 
BMD (expansion cohort only). 
Leg ulcer(s) size will be assessed in patients with leg ulcers; photographs should be taken to 
document changes. 
A 6MWT will be performed at selected sites (expansion cohort only) for NTD patients. 
An abdominal ultrasound of the spleen will be performed to evaluate spleen size for patients 
without a history of splenectomy in the dose escalation cohorts only. 
Biomarkers for iron metabolism will include serum iron, TIBC, transferrin, soluble transferrin 
receptor, ferritin, NTBI, and hepcidin. 
Biomarkers for bone metabolism will include serum CTX (a marker of bone resorption) and 
BSAP (a marker of bone formation). 
Biomarkers related to the TGF- superfamily, such as GDF15, may be tested in blood (to be 
determined).  The relationship of biomarkers to erythroid response may be investigated.  
The relationship of biomarkers to erythroid response may be investigated. 
The sponsor may request additional biomarkers for exploratory research purposes only. 


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Quality of Life tools including but not limited to the FACT-An and SF-36 questionnaires will 
assessed. 
12. 
STUDY SCHEDULE 
Please refer to Section 2 for the schedule of procedures required for each visit.  Note that all 
windows on visits should be determined relative to the date of the previous dose of ACE-536.  
12.1. 
Screening  
 Signature of the current IEC approved ICF should occur prior to initiation of any 
study-specific screening procedures. 
 All screening procedures should occur within 28 days prior to Cycle 1 Day 1. 
 Screening MRI for liver iron content (all cohorts at selected sites), MRI of the chest 
and abdomen for EMH masses (expansion cohort only at selected sites), MRI for 
spleen (if no prior splenectomy; expansion cohort only at selected sites), abdominal 
ultrasound of the spleen (if no prior splenectomy; dose escalation cohorts only) and 
ECHO, MUGA or cardiac MRI (all cohorts) can be performed up to 56 days prior to 
Cycle 1 Day 1.  If performed as part of standard of care, it does not need to be 
repeated. 
 Historical hemoglobin values will be collected for 12 weeks prior to Cycle 1 Day 1.  
Historical transfusion history will be collected for 12 months prior to Cycle 1 Day 1. 
 Baseline hemoglobin will be the average of two measurements; one performed within 
one day prior to Cycle 1 Day 1, and the other performed during the screening period 
(Day -28 to Day -1). 
 Pregnancy test (urine or serum) and menstrual history is required of female patients 
of child bearing potential only. 
 Concomitant medications taken within 28 days prior to Cycle 1 Day 1 will be 
documented in the CRF. 
 Screen failure information will be maintained to document specific information, 
including but not limited to, reason for failure. 
 For the expansion cohort patients only, administration of quality of life 
questionnaires. 
 For blood pressure measurements, if the systolic blood pressure is ≥ 150 mmHg, the 
diastolic blood pressure is ≥ 95 mmHg, and/or the absolute increase in either measure 
from baseline is ≥ 20 mmHg, perform one repeat blood pressure assessment after a 
minimum of 15 minutes. 


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12.2. 
Dosing Days and Interim Visits 
 For the first dose of ACE-536, dosing should occur after a minimum of 7 days 
post-transfusion; subsequent doses should not be given within 24 hours of transfusion 
or planned transfusion. 
 Note: For any RBC transfusions received during the study, collect hemoglobin value 
just prior to transfusion.  
 Each TD patient in the expansion cohort will have a baseline “pre-transfusion 
hemoglobin threshold” which will be calculated based on transfusion history and will 
be used for determining when to transfuse during the study 
 All screening and Cycle 1 Day 1 procedure results required to confirm eligibility 
must be obtained and reviewed prior to study drug administration.  Patient eligibility 
must be confirmed from these results.  
 A patient will be considered enrolled into the study once a multi-digit patient 
identification number has been assigned to the patient.  After assignment of the 
patient identification number and the completion of the required procedures, study 
drug administration may occur.  Note that the patient dose must be calculated based 
on the patient’s weight on the day of dosing.   
 All study procedures on each dosing day must be done prior to administration of 
study drug. 
 Patients should be observed for a minimum of 30 minutes following treatment with 
ACE-536. 
 If a dose delay is required per the patient dose modification rules (see Section 10.8.1), 
the patient will not be dosed.  The patient will return weekly for assessment of 
hematology results and adverse events until the patient is eligible to receive the next 
dose of ACE-536. 
 The hematology results can be collected up to 24 hours prior to the dosing day.  For 
Cycle 1 Day 1, the hemoglobin should be available to confirm eligibility within one 
day prior. 
 Any non-serious AEs that occur prior to dosing on Cycle 1 Day 1 should be recorded 
in the Medical History CRF. 
 All AEs that occur after dosing on Cycle 1 Day 1 should be recorded in the AE CRF. 
 On subsequent dosing days, all AEs and abnormal findings that might require 
modification of dosing (see Section 10.8) should be reviewed prior to dosing to 
ensure that the patient is still eligible to receive additional doses of ACE-536.  
 Pregnancy test (urine or serum) and menstrual history is required of female patients 
of child bearing potential only. 
 Physical exam on Cycle 1 Day 1 should include an optional evaluation of gonadal 
size of male patients. 


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 For blood pressure measurements, if the systolic blood pressure is ≥ 150 mmHg, the 
diastolic blood pressure is ≥ 95 mmHg, and/or the absolute increase in either measure 
from baseline is ≥ 20 mmHg, perform one repeat blood pressure assessment after a 
minimum of 15 minutes. 
 Leg ulcer(s) size will be assessed in patients with leg ulcers; photographs should be 
taken to document changes. 
12.3. 
Day 85 
 Day 85 (± 2 days) is the last possible study day that ACE-536 may be administered, 
regardless of the cycle. 
 For the expansion cohort patients only, administration of quality of life 
questionnaires. 
 For blood pressure measurements, if the systolic blood pressure is ≥ 150 mmHg, the 
diastolic blood pressure is ≥ 95 mmHg, and/or the absolute increase in either measure 
from baseline is ≥ 20 mmHg, perform one repeat blood pressure assessment after a 
minimum of 15 minutes. 
12.4. 
End of Treatment Visit 
 The end of treatment visit should occur approximately 28 days after the last dose of 
ACE-536.  
 Patients who discontinue treatment early should complete the end of treatment visit at 
the time of discontinuation. 
 Patients in the expansion cohort who discontinue treatment early or do not complete 
the Day 85 visit should complete the quality of life questionnaires at the end of 
treatment visit. 
 An abdominal ultrasound of the spleen will be performed for patients without a 
history of splenectomy in the dose escalation cohorts only. 
 An MRI will be performed at selected sites to evaluate liver iron content (all cohorts), 
and an MRI of the chest and abdomen for EMH mass size (expansion cohort only), 
and spleen size (expansion cohort only). 
 A DXA will be performed at selected sites to evaluate total body, lumbar spine and 
total hip BMD (expansion cohort only). 
 A 6MWT will be performed in NTD patients at selected sites (expansion cohort 
only). 
 Leg ulcer(s) size will be assessed in patients with leg ulcers; photographs should be 
taken to document changes. 
 Pregnancy test (urine or serum) and menstrual history will be collected for women of 
child bearing potential. 
 Physical exam should include an optional evaluation of gonadal size of male patients. 


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 For blood pressure measurements, if the systolic blood pressure is ≥ 150 mmHg, the 
diastolic blood pressure is ≥ 95 mmHg, and/or the absolute increase in either measure 
from baseline is ≥ 20 mmHg, perform one repeat blood pressure assessment after a 
minimum of 15 minutes. 
12.5. 
End of Study Visit 
 The end of study visit should occur approximately 28 days after the end of treatment 
visit.  
 Patients who discontinue treatment early should complete the end of study visit 
approximately 28 days after the end of treatment visit. 
 For the expansion cohort patients only, administration of quality of life 
questionnaires. 
 If a patient has a positive anti-drug antibody (ADA) result at the last visit, the patient 
may be asked to return for additional ADA testing every three months, until a 
negative result is obtained or the result is considered to be stabilized. 
 Menstrual history will be collected for women of child bearing potential; pregnancy 
test not required. 
 Physical exam should include an optional evaluation of gonadal size of male patients. 
 For blood pressure measurements, if the systolic blood pressure is ≥ 150 mmHg, the 
diastolic blood pressure is ≥ 95 mmHg, and/or the absolute increase in either measure 
from baseline is ≥ 20 mmHg, perform one repeat blood pressure assessment after a 
minimum of 15 minutes. 
 Leg ulcer(s) size will be assessed in patients with leg ulcers; photographs should be 
taken to document changes. 
12.6. 
Termination of Study 
The sponsor may terminate this study or discontinue a cohort, after consultation with the 
investigator(s), or at any time, for safety or administrative reasons.  The sponsor will terminate 
the study if the occurrence of SAEs or other findings suggests unacceptable risk to the health of 
the patients.   
 


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13. 
STUDY DRUG MATERIALS AND MANAGEMENT 
13.1. 
Study Drug 
ACE-536 is a recombinant fusion protein consisting of a modified form of the extracellular 
domain (ECD) of the human activin receptor IIB (ActRIIB) linked to the human IgG1 Fc 
domain. 
13.2. 
Study Drug Packaging and Labeling 
ACE-536 drug product is provided as a sterile, liquid formulation at a nominal concentration of 
50 mg/mL in Tris-buffered saline, pH 7.2 ± 0.5, contained in type I, 13-mm borosilicate glass 
vials (stoppered and crimp-sealed).  Each single-use vial contains 0.5 mL (25 mg) of ACE-536 
solution for injection. 
13.3. 
Study Drug Storage 
ACE-536 is stored frozen at  -65˚C until use. 
13.4. 
Study Drug Preparation 
Please refer to the Study Reference Guide, provided under separate cover, for detailed ACE-536 
drug handling, administration, and storage instructions.   
13.5. 
Study Drug Administration 
ACE-536 will be administered by SC injection.  Multiple injections may be required to 
administer the appropriate dose at higher dose levels; however, no more than 3 injections will be 
administered per dose.  Patients should be observed for a minimum of 30 minutes following 
treatment with ACE-536. 
13.6. 
Study Drug Accountability 
Accountability for ACE-536 is the responsibility of the investigator.  Investigational clinical 
supplies must be received by a designated person at the clinical site and kept in a secured 
location.  The clinical site must maintain accurate records demonstrating dates and amounts of 
ACE-536 received, to whom it was dispensed (patient-by-patient accounting), and accounts of 
any ACE-536 accidentally or deliberately destroyed or returned. 
Unless otherwise notified, all vials of ACE-536, both used and unused, must be saved for drug 
accountability purposes.  The used vials may be discarded, per the institution’s standard practice, 
after drug accountability assessment has been completed by the clinical monitor.  At the end of 
the study, the sponsor will provide direction for the outcome of all unused vials.  Following on 
the sponsor’s instructions, the investigator must either return all unused vials of ACE-536 to the 
sponsor or destroy them at the clinical site.  In either case, the outcome must be documented on 
the drug accountability log.  


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13.7. 
Study Drug Handling and Disposal 
Please refer to the Study Reference Guide, provided under separate cover, for detailed ACE-536 
drug handling, administration, storage, and disposal instructions.  
14. 
ASSESSMENT OF SAFETY 
14.1. 
Adverse Event Definitions 
Adverse Event 
An adverse event (AE) is any untoward medical occurrence in a patient or clinical investigation 
patient administered a study drug, which does not necessarily have a causal relationship with this 
treatment.  An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of the study drug 
whether or not it is considered related to the study drug.  
Abnormal laboratory and other abnormal investigational findings (e.g., physical exam, ECG) 
should not be reported as AEs unless they are associated with clinical signs and symptoms, lead 
to treatment discontinuation, or are otherwise considered clinically relevant by the investigator.  
In cases of surgical or diagnostic procedures, the condition/illness leading to such a procedure is 
considered as the AE rather than the procedure itself.  In case of a fatality, the cause of death is 
considered as the AE, and the death is considered as its outcome. 
Unexpected Adverse Events 
An unexpected AE is an AE that is not described in nature or severity in the Investigator’s 
Brochure.  
Events Not to Be Considered as Adverse Events 
Pre-existing medical conditions/signs/symptoms present before the Screening period that do not 
worsen in severity or frequency during the study are defined as Baseline Medical Conditions, 
and are not to be considered AEs.  Anticipated day-to-day fluctuations of pre-existing conditions, 
including the disease under study, that do not represent a clinically significant exacerbation or 
worsening need not be considered adverse events. 
Serious Adverse Event 
A serious adverse event (SAE) is any AE, occurring at any dose level/regimen and regardless of 
causality that: 
 Results in death. 
 Is life-threatening:  Life-threatening means that the patient was at immediate risk of 
death from the reaction as it occurred (i.e., it does not include a reaction which 
hypothetically might have caused death had it occurred in a more severe form). 
 Requires inpatient hospitalization or prolongation of existing hospitalization; 
however, a hospitalization for an elective procedure will not be considered an SAE.  
 Results in persistent or significant disability/incapacity. 


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 Is a congenital anomaly/birth defect. 
 Is an important medical event: an important medical event is an event that may not 
result in death, be life-threatening, or require hospitalization, but may be considered 
an SAE when, based upon appropriate medical judgment, it may jeopardize the 
patient and may require medical or surgical intervention to prevent one of the 
outcomes listed in the definitions for SAEs.  Examples of such medical events include 
allergic bronchospasm requiring intensive treatment in an emergency room or at 
home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, 
or the development of drug dependency or drug abuse.   
For the purposes of reporting, any suspected transmission of an infectious agent via a medicinal 
product is also considered a serious adverse reaction and all such cases should be reported in 
expedited manner. 
Events Not to Be Considered as Serious Adverse Events 
Elective hospitalizations to administer or to simplify study treatment or procedures are not 
considered SAEs.  
14.2. 
Pregnancy and In Utero Drug Exposure 
The investigator will attempt to collect pregnancy information if a female patient or a male 
patient’s female partner becomes pregnant while the patient is participating in this study.  The 
pregnancy information will be recorded on the appropriate form and must be submitted to the 
Sponsor within 2 weeks of learning of the pregnancy.  The patient or partner will be followed for 
the outcome of the pregnancy.  Information on the status of the mother and child will be 
forwarded to the Sponsor or designee.  Generally, follow up will be no longer than 6 to 8 weeks 
following the estimated delivery date.  Any premature termination of the pregnancy will be 
reported. 
14.3. 
Severity 
Investigators must evaluate the severity/intensity of AEs according to the current active minor 
version of the National Cancer Institute Common Toxicity Criteria for Adverse Events, version 
4.0 (NCI-CTCAE v4.0), preferentially using the graded scales.  If there is a change in severity of 
an adverse event, it must be recorded as a separate event.  If a particular AE’s severity/intensity 
is not specifically graded, the investigator should apply the general guidelines for determination 
of Grade 1 through Grade 5 as listed in the NCI-CTCAE v4.0 cover page (as shown below), 
using their best medical judgment: 
Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated. 
Grade 2: Moderate; minimal, local, or noninvasive intervention indicated; limiting 
age-appropriate instrumental activities of daily living (preparing meals, shopping for groceries or 
clothes, using the telephone, managing money, etc.). 
Grade 3: Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self-care activities of daily living 


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(bathing, dressing and undressing, feeding self, using the toilet, taking medications, and not 
bedridden). 
Grade 4: Life-threatening consequences; urgent intervention indicated. 
Grade 5: Death related to AE. 
14.4. 
Relationship to Study Drug 
Investigators must also assess the causal relationship of each AE to ACE-536.  Factors for the 
assessment of causal relationship include, but are not limited to, temporal relationship between 
the AE and the administration of ACE-536, known side effects of ACE-536, medical history, 
concomitant therapy, course of the underlying disease, and pertinent study procedures.  
Probable: A causal relationship is clinically/biologically highly plausible and there is a 
plausible time sequence between onset of the AE and administration of ACE-536 and 
there is a reasonable response on withdrawal. 
Possible: A causal relationship is clinically/biologically plausible and there is a plausible time 
sequence between onset of the AE and administration of ACE-536. 
Unlikely: A causal relationship is improbable and another documented cause of the AE is most 
plausible. 
Unrelated: A causal relationship can be definitively excluded and another documented cause of 
the AE is most plausible. 
14.5. 
Documentation and Methods of Reporting of Adverse Events by 
Investigator 
It is the responsibility of the Investigator to document all adverse events that occur during the 
study. Patients will be evaluated and questioned generally for AEs during the course of the study, 
starting at the signing of the informed consent.  The Investigator must report in detail all adverse 
signs and symptoms which are either volunteered by subjects or observed during or following the 
course of investigational product administration on the appropriate CRF page.  All non-serious 
AEs occurring after signing of the ICF until a patient is dosed on Cycle 1 Day 1 are to be 
documented on the medical history CRF.  All AEs and SAEs occurring after the Cycle 1 Day 1 
dose through 56 days after the last study drug administration (End of Study visit) are to be 
reported and documented on the AE CRF. 
All AEs spontaneously reported by the patient and/or in response to an open question from study 
personnel or revealed by observation, physical examination, or other diagnostic procedures will 
be recorded on the AE CRF.  Any clinically relevant changes in laboratory assessments, or other 
clinical findings as described in Section 14.1, are considered AEs and must be recorded on the 
AE CRF.  AEs are to be followed for resolution as described in Section 14.6.  
It is important that each AE report include a description of the event, duration (onset and 
resolution dates), severity, relationship with ACE-536, any other potential causal factors, any 
treatment given or other action taken (including dose modification or discontinuation of 
ACE-536) and outcome.  In addition, serious AEs (SAEs) should be identified and the 


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appropriate seriousness criteria documented.  AEs categorized as SAEs must also be documented 
using an SAE Report Form as described in Section 14.5.1. 
Specific guidance can be found in the CRF Completion Guidelines provided by the Sponsor or 
designee.  
14.5.1. 
Documentation of Serious Adverse Events 
All SAEs that occur after the first study drug administration on Cycle 1 Day 1 until 56 days after 
the last study drug administration (End of Study visit) are to be documented on the AE 
CRF.  SAEs should not be reported for patients who are considered screen failures unless the 
event is deemed due to a protocol required procedure. 
For all SAEs, an SAE form must be completed with as much information as possible and 
submitted within the time frame described in Section 14.7.   
When new significant information is obtained, as well as when the outcome of an event is 
known, the investigator should record the information on a new SAE form.  If the patient was 
hospitalized, a copy of the discharge summary must be included as part of the patient medical 
file.  In all instances, the investigator should follow up with patients until the outcome of the 
SAE is known. 
14.6. 
Reporting Period and Monitoring of Patients with Adverse Events 
As described in Section 14.5, all AEs must be recorded in the CRF up until the end of the 
treatment period.  All patients who took at least one dose of study drug, whether they completed 
the treatment period or not, should complete the end of treatment procedures.   
All AEs will be followed until clinical database lock (or resolution if it occurs before database 
lock).  All SAEs will undergo active follow up until resolved or the event becomes chronic or 
stable.  Follow up data for SAEs obtained after clinical database lock will be incorporated into 
the ACE-536 safety database. 
14.7. 
Notification about Serious Adverse Events 
If an SAE occurs during the reporting period, the investigator must immediately (i.e., within a 
maximum 24 hours after becoming aware of the event) inform the sponsor via the CRO by 
telephone, fax, or e-mail. 
All written reports should be transmitted using the study-specific SAE Report Form, which must 
be completed by the investigator following specific completion instructions.  Names, addresses, 
and telephone and fax numbers for SAE reporting are located on the SAE Report Form and in 
the completion instructions provided in the Study Manual.  When an SAE (or follow up 
information) is reported by telephone, a written report must be sent immediately thereafter by fax 
or e-mail.  Reporting procedures and timelines for follow up information are the same as for the 
initially reported SAE.  
Relevant pages from the CRF may be provided in parallel (e.g., medical history, concomitant 
therapy).  In all cases, the information provided in the SAE Report Form must be consistent with 
the data that are recorded in the corresponding sections of the CRF. 


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The investigator/reporter must respond to any request for follow up information or to any 
question the Sponsor or designee may have on the AE within the same timelines as described for 
initial reports.  This is necessary to permit a prompt assessment of the event by the Sponsor and 
(as applicable) to allow the Sponsor to meet regulatory timelines associated with expedited 
reporting obligations. 
Requests for follow up will usually be made by the responsible clinical research associate or 
Medical Monitor, or an Acceleron pharmacovigilance representative who may contact the 
investigator directly to obtain clarification on a particularly critical event. 
14.7.1. 
Safety Reporting to Health Authorities, Independent Ethics Committees, 
Institutional Review Boards, and Investigators 
The Sponsor will send appropriate safety notifications to Health Authorities in accordance with 
applicable laws and regulations. 
The investigator must comply with any applicable site-specific requirements related to the 
reporting of SAEs involving his/her patients to the IEC that approved the study. 
In accordance with International Conference on Harmonization (ICH) Good Clinical Practice 
(GCP) guidelines, the Sponsor will inform the investigator of “findings that could adversely 
affect the safety of patients, impact the conduct of the study, or alter the IEC’s 
approval/favorable opinion to continue the study.”  
The Sponsor will inform the investigator of AEs that are both serious and unexpected and are 
considered to be related to ACE-536 (“suspected unexpected serious adverse reactions” or 
SUSARs). The investigator should place copies of these Safety Reports in the Investigator Site 
File.  National regulations with regard to Safety Report notifications to investigators will be 
followed. 
When specifically required by regulations and guidelines, the Sponsor will provide appropriate 
Safety Reports directly to the concerned lead IEC and will maintain records of these 
notifications.  When direct reporting by the Sponsor is not clearly defined by national or 
site-specific regulations, the investigator will be responsible for promptly notifying the 
concerned IEC of any Safety Reports and for filing copies of all related correspondence in the 
Investigator Site File. 
For studies covered by the European Union Clinical Trials Directive 2001/20/EC, the Sponsor’s 
responsibilities regarding the reporting of SAEs/SUSARs will be carried out in accordance with 
that Directive and with the related Detailed Guidances.  
 


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15. 
STATISTICS 
15.1. 
Analysis Populations 
For all analysis populations, patients will be analyzed according to assigned cohort. 
The Intent-to-Treat (ITT) Population: All patients enrolled in the study. 
Efficacy Evaluable (EE) Population: All patients administered at least one dose of ACE-536, 
and 1) at least 2 hemoglobin values ≥ 14 days apart post-treatment, not influenced by transfusion 
in non-transfusion dependent patients, OR, 2) at least 12 weeks of transfusion frequency data 
prior to Cycle 1 Day 1 and following Cycle 1 Day 1 in transfusion dependent patients. 
Safety Population: All patients who received at least 1 dose of ACE-536. 
Pharmacokinetics Population: All patients who have received at least 1 dose of ACE-536 and 
have sufficient pharmacokinetic samples collected and assayed for PK analysis. 
15.2. 
Statistical Plan 
Summary statistics will be presented for continuous/quantitative variables, by way of number of 
patients (n), mean, standard deviation (SD), median, minimum and maximum, and by way of 
group frequencies and percentages for categories of qualitative variables.  Percentages will be 
calculated using the total patients per treatment/subgroup.  95% confidence intervals (CIs) will 
be presented as applicable. 
All patient data will be presented in separate data listings. 
15.2.1. 
Patient Accountability and Demographics 
Exposure to study drug and reasons for discontinuation of study will be tabulated, and 
demographics will be presented using descriptive statistics (i.e., mean, standard deviation, 
median, and range).   
15.2.2. 
Primary Efficacy Analysis  
The primary efficacy endpoint is defined as the proportion of β-thalassemia  patients who have 
an erythroid response, defined as 1) a hemoglobin increase of ≥ 1.5 g/dL from baseline for 
≥ 14 days (in the absence of RBC transfusions) in non-transfusion dependent patients or 2) 
≥ 20% reduction in RBC transfusion burden compared to pretreatment in transfusion dependent 
patients. 
Transfusion burden will be defined as the ratio of RBC transfusion units (or mLs) transfused 
during an interval to the duration (days) of that interval, where the interval may be during the 
pretreatment period or the treatment plus follow-up period.  The interval during the pretreatment 
period will be defined as the 12 weeks prior to Cycle 1 Day 1.  An interval during the treatment 
plus follow-up period will be defined as any 12-week interval after Cycle 1 Day 1. 
The erythroid response will be summarized using both a point estimate and its exact 95% 
confidence interval based on binomial distribution.  The primary efficacy analysis will be 
performed using the EE population. 


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No direct comparison testing with concurrent or historical controls will be performed.   
Baseline hemoglobin will be an average of 2 or more measurements; one measure performed 
within one day prior to Cycle 1 Day 1 and at least one other performed during the screening 
period (Day -28 to Day -1).  Hemoglobin measurements within 2 weeks following RBC 
transfusion will be excluded. 
15.2.3. 
Secondary Analysis 
Safety analysis: To assess clinical safety, adverse events, vital sign measurements, and clinical 
laboratory information will be summarized.  Adverse Events will be coded using the Medical 
Dictionary for Regulatory Activities (MedDRA).  Changes from baseline for clinical laboratory 
values and vital signs will be summarized over time.  Descriptive statistics and shift tables will 
be generated as appropriate. 
The secondary efficacy/pharmacodynamic analysis will be performed using the ITT and EE 
populations and will include: 
 Erythroid response, as defined above for primary endpoint, performed using the ITT 
population.  
 For non-transfusion dependent patients, the proportion of patients with an increase of 
≥ 1.0 g/dL from pretreatment hemoglobin maintained ≥ 8 weeks. 
 For non-transfusion dependent patients, the proportion of patients with an increase of 
≥ 1.5 g/dL from pretreatment hemoglobin maintained ≥ 8 weeks. 
 For transfusion dependent patients, the proportion of patients who have ≥ 50% 
reduction in RBC transfusion burden compared to pretreatment using 12-week 
intervals.  
 For transfusion dependent patients, the proportion of patients who have ≥ 50% 
reduction in RBC transfusion burden compared to pretreatment using 8-week 
intervals.  
 For transfusion dependent patients, the proportion of patients who have no RBC 
transfusions ≥ 8 weeks. 
 Time to erythroid response and duration of erythroid response. 
 Change from pretreatment in transfusion burden in transfusion dependent patients 
 Change in hemoglobin level in non-transfusion dependent patients 
 Change in pre-transfusion hemoglobin levels in transfusion dependent patients 
 Change from baseline in erythropoiesis parameters including serum erythropoietin 
levels, hemoglobin analysis (electrophoresis, globin chain RNA), reticulocytes, and 
nucleated RBCs 
 Change from baseline in hemolysis parameters include haptoglobin, indirect bilirubin, 
and lactate dehydrogenase (LDH) 
 Change from baseline in iron metabolism parameters include serum iron, total iron 
binding capacity (TIBC), transferrin, calculated transferrin saturation, soluble 


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transferrin receptor, ferritin, non-transferrin bound iron (NTBI), hepcidin, and liver 
iron content (LIC) by MRI 
 Change from baseline in bone metabolism parameters include bone specific alkaline 
phosphatase (BSAP) and C-telopeptide of type I collagen (CTX)  
 Change from baseline in spleen size (in subset of patients with no prior-splenectomy) 
The exploratory endpoints will include evaluation of biomarkers related to the TGF- 
superfamily, quality of life, change in spleen size (in subset of patients with no prior 
splenectomy), change in BMD, change in EMH mass size, change in leg ulcer size, and change 
in the 6MWT distance in NTD patients. 
All binary endpoints will be summarized using both a point estimate and its exact confidence 
interval based on the binomial distribution.  The time-to-event type secondary endpoints will be 
analyzed using Kaplan-Meier method to estimate the survival curve and median time to event 
and 95% confidence interval.   
15.2.4. 
Pharmacokinetics analysis  
Non-compartmental PK parameters for ACE-536, such as maximum plasma concentration 
(Cmax), time to maximum plasma concentration (tmax), and area under the concentration/time 
curve (AUC), will be estimated.  Dose proportionality may be assessed using the exposure data 
(eg, Cmax, AUC) after the first dose if the sufficient dose levels are studied.  Descriptive statistics 
will be provided for serum concentrations and PK parameters.  The relationship between 
ACE-536 exposure and response (ie, safety, efficacy, and biomarkers) may be explored, if 
appropriate. 
15.2.5. 
Anti-drug antibody analysis  
The results of anti-drug and neutralizing antibody testing for ACE-536 and human ActRIIB 
protein versus time will be presented.  Exploratory analysis will be performed on the potential 
effect of anti-drug antibodies on ACE-536 PK and drug exposure if anti-drug antibody tests are 
deemed positive. 
15.3. 
Determination of Sample Size 
There is no formal sample size calculation for the dose escalation portion of the study.  A sample 
size of 30 evaluable patients in the expansion cohort will provide approximately 87% power with 
1-sided significance level of 0.05 to differentiate an erythroid response rate of 30% from a 
minimal erythroid response rate of 10%. 
15.4. 
Interim Analysis 
There are no planned interim analyses.  However, safety and erythroid response data will be 
reviewed periodically throughout the study. 


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15.5. 
Deviation from Original Analysis Plan 
A formal Statistical Analysis Plan (SAP) for the analysis and presentation of data from this study 
will be prepared before the database lock.  Deviations from the statistical analyses outlined in 
this protocol will be indicated in this plan; any further modifications will be noted in the final 
clinical study report. 
16. 
 SOURCE DOCUMENTATION AND INVESTIGATOR FILES 
16.1. 
Study Monitoring 
The clinical monitor will arrange to visit the clinical sites at regular intervals during the study.  
The monitoring visits must be conducted according to the applicable ICH and GCP guidelines to 
ensure protocol adherence, quality of data, drug accountability, compliance with regulatory 
requirements, and continued adequacy of the clinical sites and their facilities.  During these 
visits, CRFs and other data related to the study will be reviewed and any discrepancies or 
omissions will be resolved.  The clinical monitor will be given access to study-relevant source 
documents (including medical records) for purposes of source data verification. 
16.2. 
Audits and Inspections 
The investigators and clinical sites will permit trial-related monitoring, audits, IEC review, and 
regulatory inspections as requested by FDA or other health authorities and the sponsor or 
designee.  In addition to CRFs, the clinical site will permit direct access to source 
data/documents (ie, original medical records, laboratory reports, hospital documents, progress 
reports, signed ICFs, etc.).  During and/or after completion of the study, quality assurance 
officers named by the sponsor or the regulatory authorities may wish to perform on-site audits.  
The investigator is expected to cooperate with any audit and provide assistance and 
documentation (including source data) as requested. 
 


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17. 
QUALITY CONTROL AND QUALITY ASSURANCE 
17.1. 
Data Quality Control and Quality Assurance 
17.1.1. 
Investigator Responsibility 
The investigator is responsible for ensuring the study is conducted according to the protocol, 
Code of Federal Regulations, GCP, and applicable regulatory requirements.  The investigator’s 
responsibilities are outlined in these documents and must include the responsibility to obtain a 
signed informed consent prior to patient participation in the study. 
17.1.2. 
Protocol Modifications 
The investigator should not modify the protocol without agreement from the sponsor and prior 
review or approval by the IEC, unless an emergency situation requires protocol modification to 
ensure the safety of patients.  Any deviations from the protocol should be documented by the 
investigator or designee.  


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18. 
CONFIDENTIALITY 
To maintain patient privacy, all CRFs, study drug accountability records, study reports and 
communications will identify the patient by the assigned patient identification number.  The 
investigator will grant clinical monitor(s) and auditor(s) from the sponsor or designee and 
regulatory authorities’ access to the patient’s original medical records for verification of data 
gathered on the CRFs and to audit the data collection process.  The patient’s confidentiality will 
be maintained and will not be made publicly available.  The patient’s medical information will 
only be released to the extent permitted by the applicable laws and regulations. 
All information regarding the investigational product supplied by the sponsor to the investigator 
is privileged and confidential information.  The investigator agrees to use this information to 
accomplish the study and will not use it for other purposes without consent from the sponsor.  It 
is understood that there is an obligation to provide the sponsor with complete data obtained 
during the study.  The information obtained from the clinical study will be used towards the 
development of the investigational product and may be disclosed to regulatory authorities, other 
investigators, corporate partners, or consultants as required. 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by United States Law.  This Web site will not include information that can identify patients.  At 
most, the web site may include a summary of the results.  
Publication Policy 
All information concerning ACE-536 is considered confidential and shall remain the sole 
property of the sponsor.  The investigator(s) agree to use this information only in conducting the 
study and shall not use it for any other purposes without the sponsor’s written approval.  The 
investigator(s) agree not to disclose the sponsor’s confidential information to anyone except to 
persons involved in the study that need such information to assist in conducting the study, and 
then only on like terms of confidentiality and non-use. 
It is understood by the investigator(s) that the information developed from this clinical study will 
be used by the sponsor in connection with the development of ACE-536, and therefore may be 
disclosed as required to regulatory agencies.  To allow for the use of the information derived 
from clinical studies, it is understood that there is an obligation to provide the sponsor with 
complete test results and all data developed in the study. 
No publication or disclosure of study results will be permitted except as specified in a separate, 
written, agreement between the sponsor and the investigator(s). 
19. 
PROTOCOL AMENDMENTS 
Protocol amendments that impact patient safety, change the scope of the investigation, or affect 
the scientific quality of the study must be approved by the IEC and submitted to the appropriate 
regulatory authorities before implementation. 
In the event that the protocol needs to be modified immediately to eliminate an apparent hazard 
to a patient, the sponsor will implement the protocol change and subsequently amend the 
protocol and notify the regulatory authorities and/or the IEC, as appropriate. 


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20. 
DATA HANDLING AND RECORDKEEPING 
20.1. 
Case Report Form Completion 
Case report forms will be completed for each enrolled patient.  It is the investigator’s 
responsibility to ensure the accuracy, completeness, and timeliness of the data reported in the 
patient’s CRF.  Source documentation supporting the CRF data should indicate the patient’s 
participation in the study and should document the dates and details of study procedures, AEs, 
and patient status. 
Investigators will maintain copies of the CRFs at the clinical site.  For patients who discontinue 
or terminate from the study, the CRFs will be completed as much as possible, and the reason for 
the discontinuation or termination clearly and concisely specified on the appropriate CRF. 
20.2. 
Retention of Records 
The investigator will maintain all study records according to ICH-GCP and applicable regulatory 
requirements.  Records will be retained for at least 2 years after the last marketing application 
approval or 2 years after formal discontinuation of the clinical development of the investigational 
product, or according to applicable regulatory requirements.  If the investigator withdraws from 
the responsibility of keeping the study records, custody must be transferred to a person willing to 
accept the responsibility.  The sponsor must be notified in writing if a custodial change occurs. 


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21. 
STUDY FINANCE AND INSURANCE 
21.1. 
Study Finance 
The costs necessary to perform the study will be agreed with each Investigator and will be 
documented in a separate financial agreement that will be signed by the Investigator and 
Acceleron Pharma Inc. or designee, prior to the study commencing.  
21.2. 
Insurance 
The Sponsor has insurance coverage for study-related ACE-536 induced injury and other 
liabilities incurred during clinical studies which will provide compensation for any study-related 
injury according to the guidelines set out by the Association of the British Pharmaceutical 
Industry (ABPI), namely “Clinical Studies Compensation for Medicine Induced Injury”. 


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22. 
REFERENCES 
1. Massague J. TGF-beta signal transduction. Annu Rev Biochem 1998;67:753-91. 
2. Centis F, Tabellini L, Lucarelli G, et al. The importance of erythroid expansion in 
determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia 
major. Blood 2000;96:3624-9. 
3. Testa U. Apoptotic mechanisms in the control of erythropoiesis. Leukemia 2004;18:1176-99. 
4. Rivella S. Ineffective erythropoiesis and thalassemias. Curr Opin Hematol 2009;16:187-94. 
5. Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service 
indicators. Bull World Health Organ 2008;86:480-7. 
6. Olivieri N, Weatherall DJ. Clinical aspects of b-thalassemia and related disorders. In: al 
MHSe, ed. Disorders of Hemoglobin. 2nd ed. Cambridge, UK: Cambridge University Press; 
2009:357. 
7. Weatherall DJ, O. A, al FSe. Inherited disorders of hemoglobin. In: al DTJe, ed. Disease 
Control Priorities in Developing Countries. 2nd ed. New York: Oxfor University Press and the 
World Bank; 2006:663. 
8. Skow LC, Burkhart BA, Johnson FM, et al. A mouse model for beta-thalassemia. Cell 
1983;34:1043-52. 
9. American Heart Association Classes of Heart Failure. 2011. (Accessed at 
http://www.heart.org/HEARTORG/Conditions/HeartFailure/AboutHeartFailure/Classes-of-
Heart-Failure UCM 306328 Article.jsp.) 
10. Yellen SB, Cella DF, Webster K, et al. Measuring fatigue and other anemia-related 
symptoms with the Functional Assessment of Cancer Therapy (FACT) measurement system. 
J Pain Symptom Manage 1997;13(2): 63-74. 
11. Cella D, Eaton D, Lai JS, et al. Combining anchor and distribution-based methods to derive 
minimal clinically important differences on the Functional Assessment of Cancer Therapy 
(FACT) anemia and fatigue scales. J Pain Symptom Manage 2002; 24(6):547-561. 
12. Eremenco SL, Cella DF, Arnold BJ. A comprehensive method for the translation and 
cross-cultural validation of health-status questionnaires. Evaluation and the Health Professions 
2005; 28(2):212-232. 
 
 


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23. 
APPENDICES 
23.1. 
Appendix 1: New York Heart Association Classification - The Stages 
of Heart Failure9 
Class 1 – Class 1 heart failure – patients with cardiac disease but resulting in no limitation of 
physical activity.  Ordinary physical activity does not cause undue fatigue, palpitation, dyspnea, 
or anginal pain.  
Class 2 – Class 2 heart failure – patients with cardiac disease resulting in slight limitation of 
physical activity.  They are comfortable at rest.  Ordinary physical activity results in fatigue, 
palpitation, dyspnea, or anginal pain.  
Class 3 – Class 3 heart failure – patients with cardiac disease resulting in marked limitation of 
physical activity.  They are comfortable at rest.  Less than ordinary activity causes fatigue, 
palpitation, dyspnea, or anginal pain.  
Class 4 – Class 4 heart failure – patients with cardiac disease resulting in inability to carry on 
any physical activity without discomfort.  Symptoms of heart failure or the anginal syndrome 
may be present even at rest.  If any physical activity is undertaken, discomfort increases.  
 


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23.2. 
Appendix 2: National Cancer Institute (NCI) Common Terminology 
Criteria for Adverse Events (CTCAE)  
See http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE 

SAP_001.pdf: 
Official Protocol Title: 
NCT number: 
Document Date: 
A Phase 2, Open-Label, Ascending Dose Study to Evaluate the 
Effects of ACE-536 in Patients with β-Thalassemia
04 August 2017
NCT01749540


 
 
 
 
 
Statistical Analysis Plan 
 
 
 
A Phase 2, Open-Label, Ascending Dose Study to Evaluate 
the Effects of ACE-536 in Patients with β-Thalassemia  
 
 
Sponsor: 
Acceleron Pharma Inc. 
128 Sidney Street 
Cambridge, MA 02139 
Tel: 
  
Investigational Product:  
Luspatercept (ACE-536) 
Protocol Number: 
A536-04 
EudraCT Number: 
2012-002499-15 
Version Number: 
1 (Final) 
Date:  
04 August 2017 
 
 
 
 
 
 
 
CONFIDENTIALITY STATEMENT 
This document contains confidential information, which should not be copied, referred to, released, or 
published without written approval from Acceleron Pharma Inc.  Investigators are cautioned that the 
information given in this brochure might be subject to change and revision.  Any conclusion regarding 
efficacy and safety must be considered provisional. 
 
PPD




A536-04 
 
Statistical Analysis Plan 
Confidential and Proprietary 
3 
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TABLE OF CONTENTS 
1. 
INTRODUCTION ..................................................................................... 5 
2. 
STUDY OBJECTIVES ............................................................................. 5 
3. 
OVERALL STUDY DESIGN ................................................................... 6 
3.1. 
Study Design .............................................................................................. 6 
3.2. 
Treatment Discontinuation ........................................................................ 7 
3.3. 
Rollover to Extension study ....................................................................... 8 
3.4. 
Sample Size ............................................................................................... 8 
4. 
ANALYSIS POPULATIONS ................................................................... 8 
4.1. 
Intent-to-Treat (ITT) Population ................................................................ 8 
4.2. 
Safety Population ....................................................................................... 8 
4.3. 
Pharmacokinetics (PK) Population ............................................................ 8 
5. 
STATISTICAL METHODOLOGY .......................................................... 9 
5.1. 
Definitions ................................................................................................. 9 
5.2. 
General Considerations .............................................................................. 9 
5.3. 
Disposition of Patients ............................................................................. 10 
5.4. 
Demographic, Baseline Characteristics, and Disease History ................. 10 
5.5. 
Study Drug Exposure ............................................................................... 10 
5.6. 
Prior and Concomitant Medication and Procedures ................................ 11 
5.6.1. 
Prior and Concomitant Medication .......................................................... 11 
5.6.2. 
RBC Transfusions .................................................................................... 11 
5.6.3. 
Non-Medication Procedures .................................................................... 11 
5.7. 
Efficacy Analyses .................................................................................... 11 
5.7.1. 
Primary Efficacy Endpoint ...................................................................... 12 
5.7.2. 
Secondary Efficacy Endpoints ................................................................. 12 
5.7.2.1. 
Modified Erythroid Response .................................................................. 12 
5.7.2.2. 
Time to and Duration of Erythroid Response .......................................... 13 
5.7.2.3. 
Iron Parameter Related Analysis ............................................................. 13 
5.7.2.4. 
Erythropoiesis Parameters ....................................................................... 13 
5.7.2.5. 
Bone Metabolism Parameters .................................................................. 14 
5.7.2.6. 
Hemolysis Parameters ............................................................................. 14 
5.7.3. 
Exploratory Endpoints ............................................................................. 14 
5.7.3.1. 
Biomarkers Related to TGF- ................................................................. 14 
5.7.3.2. 
QoL Questionnaire ................................................................................... 14 
5.7.3.3. 
Other Analysis ......................................................................................... 14 


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Statistical Analysis Plan 
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5.7.4. 
Subgroup Analysis ................................................................................... 15 
5.8. 
Safety Analysis ........................................................................................ 15 
5.8.1. 
Dose Limiting Toxicities ......................................................................... 15 
5.8.2. 
Adverse Events ........................................................................................ 15 
5.8.3. 
Laboratory Evaluations ............................................................................ 16 
5.8.4. 
Vital Signs ............................................................................................... 17 
5.8.5. 
ECG Results............................................................................................. 17 
5.8.6. 
Physical Examination .............................................................................. 17 
5.9. 
Pharmacokinetics Analysis ...................................................................... 18 
5.9.1. 
Pharmacokinetic Sampling Schedule ...................................................... 18 
5.9.2. 
Data Handling .......................................................................................... 18 
5.9.3. 
Noncompartmental Pharmacokinetic Analysis ........................................ 19 
5.9.4. 
Compartmental Pharmacokinetic Analysis .............................................. 19 
5.9.5. 
Statistical Analysis ................................................................................... 20 
5.10. 
Interim Analysis ....................................................................................... 20 
5.11. 
Protocol Deviations ................................................................................. 20 
5.12. 
Handling of Missing Data ........................................................................ 21 
5.12.1. 
Missing Dates for Adverse Event ............................................................ 21 
5.12.2. 
Missing Dates for Concomitant Medication ............................................ 21 
5.12.3. 
Missing Dates for Disease Diagnosis Date .............................................. 22 
5.13. 
Changes in Conduct or Planned Analyses from the Protocol .................. 22 
6. 
REFERENCES ........................................................................................ 22 
7. 
APPENDICES ......................................................................................... 22 
7.1. 
FACT-An Scoring Algorithm .................................................................. 22 
7.2. 
SF-36 Scoring Algorithm ........................................................................ 23 
8. 
LIST OF ABBREVIATIONS.................................................................. 27 
9. 
SCHEDULE OF EVENTS ...................................................................... 29 
 
 
 


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1. 
INTRODUCTION  
This statistical analysis plan (SAP) describes the statistical methods to be used for the 
analysis of Acceleron Protocol A536-04.  This SAP should be read in conjunction with the 
study protocol and electronic case report form (eCRF).  This version of the plan has been 
developed using the protocol amendment 04 dated 07NOV2014 and eCRF dated 
04 April 2014.  Any further changes to the protocol or eCRF may necessitate updates to the 
SAP. 
The SAP will be signed off before the study database lock.  Any deviations from the SAP 
will be described and justified in the final clinical study report (CSR). 
2. 
STUDY OBJECTIVES 
Primary Objective: 
 To evaluate the proportion of β-thalassemia patients who have an erythroid response, 
defined as: 1) a hemoglobin increase of ≥ 1.5 g/dL from baseline for ≥ 14 days (in the 
absence of red blood cell [RBC] transfusions) in non-transfusion dependent patients, or 
2) ≥ 20% reduction in RBC transfusion burden compared to pretreatment in transfusion 
dependent patients 
Secondary Objectives: 
 To evaluate safety and tolerability of ACE-536   
 To evaluate the proportion of transfusion dependent patients who have ≥ 50% 
reduction of RBC transfusion burden compared to pretreatment  
 To evaluate the time to erythroid response and duration of erythroid response  
 To evaluate the change in hemoglobin level in non-transfusion dependent patients  
 To evaluate the change in pre-transfusion hemoglobin levels in transfusion 
dependent patients  
 To evaluate changes in biomarkers of erythropoiesis, hemolysis, iron metabolism 
and bone metabolism  
 To examine the pharmacokinetic (PK) profile of ACE-536 in patients with 
β-thalassemia  
Exploratory Objectives: 
 To evaluate biomarkers related to the TGF- superfamily  
 To evaluate self-reported quality of life using the FACT-An and SF-36 
questionnaires (expansion cohort only)  
 To evaluate change in spleen size by MRI 
 To evaluate change in bone mineral density (BMD) by DXA 
 To evaluate change in extramedullary hematopoiesis (EMH) mass size by MRI 
 To evaluate change in leg ulcer size  
 To evaluate change in the 6-minute walk test (6MWT) distance in NTD patients  


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3. 
OVERALL STUDY DESIGN 
This is a phase 2, open-label, ascending dose study to evaluate the effects of ACE-536 in 
patients with β-thalassemia. 
3.1. 
Study Design 
Patients who meet the study eligibility criteria were enrolled within 28 days of screening.  
Patients in all cohorts received ACE 536, administered subcutaneously (SC), every 3 weeks 
for up to 5 cycles.  ACE-536 could not be administered after study day 85 (± 2 days).  Dose 
delay(s) and dose reduction(s) might be required for individual patients as outlined in the 
protocol.   
Each dose escalation cohort consisted of up to 6 patients.  The dose level of ACE-536 for the 
first cohort was 0.2 mg/kg and the dose level(s) for subsequent cohort(s) followed a modified 
Fibonacci dose escalation scheme (i.e., maximum dose of 0.4 mg/kg for cohort 2, 0.6 mg/kg 
for cohort 3, 0.8 mg/kg for cohort 4, 1.0 mg/kg for cohort 5, etc.).  Once a minimum of 
3 patients in a cohort had completed Study Day 29, a Safety Review Team (SRT) reviewed 
preliminary safety and hematologic response data and made recommendations to the Sponsor 
regarding whether or not to enroll an additional cohort at a higher or lower dose, complete 
enrollment of 6 patients at the current dose level, or proceed to the expansion cohort.   
The expansion cohort (n=30) was treated with ACE-536 at a starting dose level not to exceed 
the maximum dose level for the previous cohorts.  The expansion cohort included a minimum 
of 12 patients who are non-transfusion dependent (NTD) and a minimum of 12 patients who 
are transfusion dependent (TD) (including a minimum of 6 thalassemia major patients with 
onset of regular transfusions before age 4 years, and a minimum of 6 patients with onset of 
regular transfusions onset after age 4 years).  For subsequent cycles in the expansion cohort, a 
26 
patient’s dose level might be titrated from the previous dose administered for that patient.  
The maximum dose level given to a patient did not exceed the maximum dose level evaluated 
in the dose escalation cohorts.  Patients were treated with up to 5 doses of ACE 536.  
Each TD patient in the expansion cohort had a defined “pre-transfusion hemoglobin 
threshold” which was calculated based on transfusion history and was used for determining 
when to transfuse during the study.  The baseline pre transfusion hemoglobin threshold was 
the mean of all documented pre-transfusion hemoglobin values during the 12 weeks prior to 
C1D1.  During treatment, if the pre-transfusion hemoglobin value was increased by ≥ 1 g/dL 
compared to the baseline pre-transfusion hemoglobin threshold for that patient, transfusion 
should be delayed by a minimum of 7 days and/or the number of units transfused should be 
reduced by 1 or more RBC units.  Patients may be transfused at the investigator’s discretion 
for symptoms related to anemia or other requirements (e.g., infection). 
 
 


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Cohorta 
ACE-536 Dose Levelb 
(mg/kg) 
Number of Patients  
1 
0.2 
6 
2 
0.4 
6 
3 
0.6 
6 
4 
0.8 
6 
5 
1.0 
6 
6 
1.25 
5 
Expansion 
0.8 
30c 
Total (All Cohorts): 
64 
a Cohort escalation was based on SRT review and recommendation to enroll additional cohorts and/or the expansion cohort. 
b The ACE-536 dose level for cohort 1 was 0.2 mg/kg.  The dose level indicated in the table for all subsequent cohorts was 
the maximum dose level that could be recommended by the SRT for escalation per the modified Fibonacci dose escalation 
scheme. 
c The expansion cohort consisted of 30 patients, including a minimum of 12 patients who were non-transfusion dependent and 
a minimum of 12 patients who were transfusion dependent (including a minimum of 6 thalassemia major patients with onset 
of regular transfusions before age 4 years, and a minimum of 6 patients with onset of regular transfusions after age 4 years). 
3.2. 
Treatment Discontinuation 
Patients were informed that they have the right to withdraw from the study at any time for any 
reason without prejudice to their medical care.   
A patient may be discontinued from treatment for any of the following reasons:  
 Patient’s request 
 Patient’s unwillingness or inability to comply with the protocol 
 Pregnancy 
 Use of prohibited medication (e.g., hydroxyurea) 
 Medical reason or adverse event, at the discretion of the investigator and/or the 
medical monitor 
 Hypersensitivity reaction to study drug 
 At the discretion of the sponsor (e.g., termination of the study or a dose level) 
A patient may be withdrawn from the study for any of the following reasons: 
 Patient’s request 
 Patient’s unwillingness or inability to comply with the protocol 
 Death 
 Loss to follow-up 
 At the discretion of the sponsor (e.g., termination of the study) 
Patients who discontinued treatment early completed the end of treatment (EOT) follow-up 
visit at the time of discontinuation and then completed the end of study (EOS) follow-up visit 
approximately 28 days later.   


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3.3. 
Rollover to Extension Study 
Consenting patients that meet the A536-06 eligibility criteria may immediately roll over from 
A536-04 to study A536-06 following the last ACE-536 dose.  These patients may forego the 
End of Study (EOS) visit in study A536-04 to begin study A536-06.   
Patients who complete the EOS visit for study A536-04 and are ≥ 28 days post EOS visit may 
also roll over to study A536-06.  These patients will be re-assessed for eligibility by meeting 
all eligibility criteria plus additional inclusion criteria for study A536-06.  
Patients who do not meet the above criteria (e.g., ˃ 35 days after last dose in study A536-04 
and < 28 days post EOS visit in study A536-04) may still participate, but should not begin 
study A536-06 C1D1 until they have reached ≥ 28 days po st EOS visit from study A536-04. 
3.4. 
Sample Size 
There was no formal sample size calculation for the dose escalation portion of the study.  A 
sample size of 30 evaluable patients in the expansion cohort will provide approximately 87% 
power with 1-sided significance level of 0.05 to differentiate an erythroid response rate of 30% 
from a minimal erythroid response rate of 10%. 
4. 
ANALYSIS POPULATIONS 
For all analysis populations, patients will be analyzed according to assigned cohort. 
4.1. 
Intent-to-Treat (ITT) Population 
The intent-to-treat (ITT) population will consist of all enrolled patients.  This population will 
be used for all efficacy analysis.  
4.2. 
Safety Population 
Safety population will include all patients who received at least 1 dose of ACE-536.  This 
population will be used for all safety analysis. 
4.3. 
Pharmacokinetics (PK) Population 
Pharmacokinetics population will include all patients who have received at least 1 dose of 
ACE-536 and have sufficient pharmacokinetic samples collected and assayed for PK analysis 
 
 


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5. 
STATISTICAL METHODOLOGY 
5.1. 
Definitions 
Transfusion Dependent (TD)  
Based on protocol, TD patients are defined as those who required ≥ 4 units of RBCs every 8 
weeks (confirmed over 6 months prior to Cycle 1 Day 1).  
For programming implementation, a patient is defined as TD if the total RBCs is ≥ 11 units 
during 26 weeks on or prior to Cycle 1 Day 1.  
Non-Transfusion dependent (NTD)  
NTD patients are those who do not meet the above definition for TD.  For programming 
implementation, NTD patients are defined as those who received < 11 units of RBCs during 
26 weeks on or prior to Cycle 1 Day 1.  
Baseline Hemoglobin  
Baseline hemoglobin will be an average of hemoglobin measurements within 28 days of Cycle 
1 Day 1, excluding measurements within 14 days following RBC transfusion. 
Baseline Transfusion Burden   
Baseline transfusion burden will be calculated as the total amount of RBC transfusions during 
the 12 weeks on or prior to Cycle 1 day 1.  
Baseline for Other Parameters   
Baseline erythropoietin (EPO) is defined as the maximum test value within 28 days of Cycle 1 
Day 1. For all other parameters, baseline is defined as the last observation on or prior to Cycle 
1 Day 1.  
Transfusion Unit Conversion   
The transfusion amount measured in unit “mL” will be converted to “units” according to 
medical review.  
End of Treatment (EOT)   
Procedures and evaluations for the end of treatment visit should be performed 28 days 
(±7 days) after the last dose of ACE-536.   
End of Study (EOS)   
Procedures and evaluations for the end of study visit should be performed 28 days (±7 days) 
after the Day 113/EOT visit (56 days after the last dose of ACE-536). 
Combined Dose Groups  
Low dose group includes patients on 0.2mg/kg and 0.4 mg/kg dose cohorts.  High dose group 
include patients with dose level 0.6 mg/kg and above.  Suitable summaries may be presented 
by low/high dose as specified.   
5.2. 
General Considerations   
Unless otherwise noted, continuous data will be summarized with the following descriptive 
statistics: number of observations (n), mean, standard deviation (STD), minimum, median, and 
maximum.  Categorical data will be summarized with frequencies (n) and percentages (%).  In 
cases where missing data cause percentages not to sum to 100, a missing data row will be 


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provided.  Percentages will use column totals as the denominator unless otherwise indicated. 
For time to event variables, the Kaplan-Meir curves will be presented if the number of patients 
is more than 5. 
All study data will be included in study data listings.  Missing data will generally be treated as 
missing, not imputed, unless otherwise stated. 
All summaries will be presented by dose cohort, and may also be presented by low/high dose 
groups as specified.  The summaries may also be presented for TD and NTD patients separately 
as specified.  All summaries will be descriptive. No formal hypothesis testing is planned. 
5.3. 
Disposition of Patients 
The number and percentage of patients receiving study treatment who completed the treatment 
period and study period along with the associated reasons for discontinuation from treatment 
and/or withdrawal from study will be presented by dose cohort.  
5.4. 
Demographic, Baseline Characteristics, and Disease History 
The following baseline and demographic characteristics will be summarised by descriptive 
statistics by dose cohort for ITT population as well as for TD and NTD patients separately:  
 Race, ethnicity, age, gender, height, weight  
 Splenectomy (yes or no), iron chelation therapy (yes or no), prior β-thalassemia 
treatment (yes or no) 
 Baseline haemoglobin for NTD patients only and baseline transfusion burden for TD 
patients only.  
 Baseline LIC and serum Ferritin 
Demographic and baseline data, medical history, and disease history data will be listed for each 
patient. 
5.5. 
Study Drug Exposure 
Study drug exposure will be descriptively summarized by dose cohort for safety population 
and will present the duration of exposure, the number of treatment cycles, the total dose 
administered, the number of patients with dose delay and reduction, and the number of 
patients with dose increase (for expansion cohort only).  
The duration of exposure will be calculated as (last dose date – first dose date) + 21. 
The total number of cycles will be summarized by presenting the number and percentage of 
patients in each category. 
The total dose administered is the total amount of doses a patient received during the 
treatment period. 
The number of patients experiencing dose delay and reduction will be summarized by dose 
cohort.  For the expansion cohort, the number of patients with dose increase will also be 
summarized. 
Study drug administration details will be listed for each patient. 


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5.6. 
Prior and Concomitant Medication and Procedures 
5.6.1. 
Prior and Concomitant Medication 
The prior and concomitant medications are coded with WHO- DDB2E.  The medications will 
be summarized by dose group for the ITT population.  
Medications will be assigned as prior or concomitant based on the following rules: 
 If both the start and stop date exist and are before the first dose date of study drug, the 
medication will be counted as prior. 
 If the start date is on or after the first dose date of study drug, the medication will be 
counted as concomitant. 
 If the start date is before the first dose date of study drug and the stop date is after the 
first dose date of study drug or the medication is ongoing, the medication will be 
counted as prior and concomitant. 
 If the start date is missing and the stop date is before the first dose of study drug, the 
medication will be counted as prior. 
 If the start date is missing and the stop date is after the first dose of study drug or the 
medication is ongoing, the medication will be counted as concomitant. 
 If the start and stop dates are missing, the medication will be counted as concomitant. 
All prior and concomitant medications will be listed for each patient. 
5.6.2. 
RBC Transfusions  
The complete RBC transfusion records prior to and during treatment will be listed for each 
patient.  The hemoglobin values prior to transfusion will also be listed. 
5.6.3. 
Non-Medication Procedures  
Non-medication procedures will be coded using MedDRA Version 15.1 or higher.  All 
non-medication procedures will be listed for each patient. 
5.7. 
Efficacy Analyses 
All efficacy endpoint will be performed using the ITT population.  
All efficacy summaries will be presented by dose cohort and for NTD and TD patients 
separately.  In addition, the dose cohorts will be combined and presented as low dose group 
and high dose group.  Low dose group includes patients on 0.2 and 0.4 mg/kg dose cohorts. 
High dose group includes patients on 0.6 mg/kg and above.  No formal hypothesis testing is 
planned.  
In general, below rules apply to the derivations of efficacy endpoints which utilize hemoglobin 
and RBC transfusion data unless specified otherwise: 
 Hemoglobin measurements within 14 days following RBC transfusion will be 
excluded from efficacy analysis 
 For each patient, all efficacy endpoints will be derived based on an analysis cut-
off day, defined as the last dose + 56 days or the last date from transfusion record 
data, whichever is earlier.  


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5.7.1. 
Primary Efficacy Endpoint 
The primary efficacy endpoint is the proportion of β-thalassemia patients who have an 
erythroid response, defined as below:  
 For non-transfusion dependent (NTD) patients, a hemoglobin increase of ≥ 1.5 
g/dL from baseline sustained for ≥ 14 days (in the absence of RBC transfusions). 
Hemoglobin measurements within 14 days following RBC transfusion or after 56 
days of last dose will be excluded from analysis.  
 For transfusion dependent (TD) patients, a ≥ 20% reduction in RBC transfusion 
burden during any rolling 12-week window on treatment compared to baseline. 
The rolling 12-week window starts on Day 1 and increments by 1 day, e.g., from 
day 1 to day 84, day 2 to day 85, day 3 to day 86, etc.  The search window ends on 
last dose day + 56 days or last day of hemoglobin measurement, whichever is 
earlier.  
A point estimate of the proportion of patients having a response will be presented along with 
its associated exact 95% confidence interval based on binomial distribution for NTD and TD 
patients separately.   
The complete hemoglobin measurements and RBC transfusion records will be listed for each 
patient. 
5.7.2. 
Secondary Efficacy Endpoints 
5.7.2.1. 
Modified Erythroid Response 
Modified erythroid response rates will be defined as below for NTD and TD patients. 
For non-transfusion dependent (NTD) patients:  
 The proportion of patients with a mean increase of ≥ 1.0 g/dL from pretreatment 
hemoglobin during any rolling 8 weeks. 
 The proportion of patients with a mean increase of ≥ 1.5 g/dL from pretreatment 
hemoglobin during any rolling 8 weeks. 
 The proportion of patients with a mean increase of ≥ 1.0 g/dL from pretreatment 
hemoglobin during any rolling 12 weeks 
 The proportion of patients with a mean increase of ≥ 1.5 g/dL from pretreatment 
hemoglobin during any rolling 12 weeks 
For transfusion dependent (TD) patients: 
 The proportion of patients who have ≥ 50% reduction in RBC transfusion burden 
compared to pretreatment using 12-week intervals.  
 The proportion of patients who have ≥ 50% reduction in RBC transfusion burden 
compared to pretreatment using 8-week intervals.  
 The proportion of patients who have no RBC transfusions ≥ 8 weeks. 
For each of the above defined modified erythroid response rate, a point estimate will be 
presented along with the exact 95% confidence interval based on binomial distribution. 


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5.7.2.2. 
Time to and Duration of Erythroid Response  
Time to and duration of erythroid response will be analyzed for erythroid responders defined 
as below: 
 For NTD patients, mean increase of ≥ 1.0 g/dL from pretreatment hemoglobin 
during any rolling 12 weeks 
 For TD patients, ≥ 50% reduction in RBC transfusion burden compared to 
pretreatment using 12-week intervals. 
Time to erythroid response will be defined as the time from the first dose date to the first date 
of any rolling 12-week window achieving above erythroid response.  
Duration of erythroid response will be defined as the time from the starting date of the first 
rolling 12-week window achieving response to the last date of the consecutive rolling 
12-week window achieving response.  When there are multiple disjoint intervals with 
response, the longest interval will be used.  Patients with response ongoing by the analysis 
cutoff day will be censored. 
Both time to and duration of erythroid response will be analyzed as continuous variables and 
summarized by descriptive statistics. In addition, duration of erythroid response will also be 
analyzed as a time to event endpoint.  
The derivations of time to and duration of erythroid response will be listed for each patient. 
5.7.2.3. 
Iron Parameter Related Analysis 
Iron metabolism parameters including serum iron, total iron binding capacity (TIBC), 
transferrin, soluble transferrin receptor, ferritin, non-transferrin bound iron (NTBI), and 
hepcidin will be taken at Screening, Day 1, 8, and 15 of Cycles 1, Day 1 of Cycle 2, 3, 4, 5, 
EOT and EOS visits.  Descriptive statistics of actual values, absolute and percentage change 
from baseline values at each postbaseline time point will be presented by dose cohort.  The 
summary will also be provided by NTD and TD. 
MRI for Liver Iron Content (LIC) will be performed at baseline and EOT.  Descriptive 
statistics of actual values, absolute and percentage change from baseline at EOT visit will be 
presented by dose cohort.  The summary will be done by NTD/TD and baseline LIC ≥ 3 and 
< 3 mg/g dw subgroups separately.  
LIC response will be defined as LIC reduction ≥ 1 mg/g dw at EOT compared with baseline, 
for patients with baseline LIC ≥ 3 mg/g dw only.  LIC response rate along with the exact 95% 
confidence interval will be estimated for NTD and TD patients respectively, as well as by 
baseline ICT use (yes vs no).  
Shift analyses will also be performed to summarize serum ferritin EOT change from baseline. 
Serum ferritin will be grouped into three categories: < 300, ≥ 300 and ≤ 1000, ≥ 1000 (µg/L). 
Plots of iron parameters for both mean observed values and mean change from baseline over 
time will be presented by dose cohort. 
5.7.2.4. 
Erythropoiesis Parameters 
Blood samples relating to erythropoiesis parameters include serum erythropoietin (EPO) 
levels, hemoglobin electrophoresis, globin chain RNA, reticulocytes and nucleated RBCs.  
EPO will be taken at Screening, Day 1 of Cycles 1, 2, 4, EOT, and EOS.  Hemoglobin 
electrophoresis and globin chain RNA will be taken at C1D1, C4D1 and EOT.  Reticulocytes 


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and nucleated RBCs will be taken at Screening, Day 1, 8, and 15 of Cycles 1, 2, 3, 4, 5, EOT 
and EOS visits.  Descriptive statistics of observed values, absolute and percentage change 
from baseline values at each postbaseline visit will be presented by dose cohort and for TD 
and NTD separately.  Plots of erythropoiesis parameters for both mean observed values and 
mean percentage change from baseline over time will be presented by dose cohort. 
5.7.2.5. 
Bone Metabolism Parameters 
Bone metabolism parameters including bone specific alkaline phosphatase (BSAP) and 
C-telopeptide of type I collagen (CTX) will be taken at Cycle 1 Day 1, Cycle 4 Day 1, and 
EOT visits.  Descriptive statistics of observed values, absolute and percentage change from 
baseline values at each postbaseline visit will be presented by dose cohort.  
5.7.2.6. 
Hemolysis Parameters 
Hemolysis parameters include haptoglobin, indirect bilirubin, and lactate dehydrogenase 
(LDH).  Haptoglobin will be taken at Screening, Day 1, 8, and 15 of Cycles 1, 2, 3, 4, 5, EOT 
and EOS visits. Indirect bilirubin and LDH will be taken at Screening, Day 1 and 15 of Cycle 
1, Day 1 of Cycles 2, 3, 4, 5, EOT and EOS visits.  Descriptive statistics of observed values, 
absolute and percentage change from baseline values at each postbaseline visit will be 
presented by dose cohort.  Plots of hemolysis parameters for both mean observed values and 
mean percentage change from baseline over time will be presented by dose cohort and for TD 
and NTD separately. 
5.7.3. 
Exploratory Endpoints 
5.7.3.1. 
Biomarkers Related to TGF-  
For biomarkers GDF8, GDF11, and GDF15, descriptive statistics of observed values at 
baseline, observed values, changes and percentage changes from baseline at each 
postbaseline visit will be presented by dose cohort.  
5.7.3.2. 
QoL Questionnaire 
QoL questionnaires including FACT-An and SF-36 will be completed at Screening, Cycle 5 
Day 1 or End of Treatment (for patients who withdraw early) visits for the Expansion Cohort 
only.  The scoring algorithms for FACT-An and SF-36 are included in the appendix. 
Individual subscale scores and total scores will be summarised by visit.  All derived subscale 
and total scores along with all individual responses will be listed for each patient.  
5.7.3.3. 
Other Analysis 
The following additional exploratory endpoints will be presented in listings only:  
 Change in spleen size by MRI (in subset of patients with no prior-splenectomy) 
 Change in bone mineral density (BMD) by DXA 
 Change in extramedullary hematopoiesis (EMH) mass size by MRI  
 Change in leg ulcer size (area) 
 Change in the 6-minute walk test (6MWT) distance in NTD patients 
 Hormone parameters (including estradiol, free testosterone, and total testosterone) 


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For the following continuous variables, the descriptive statistics including mean, median and 
range will be presented by dose cohort:  
 Change from pretreatment in transfusion burden in transfusion dependent patients 
 Change in hemoglobin level in non-transfusion dependent patients 
 Change in pre-transfusion hemoglobin levels in transfusion dependent patients 
5.7.4. 
Subgroup Analysis 
Subgroup analyses of erythroid response will be performed with the following baseline 
factors, for NTD and TD patients separately: 
 Age (< 32 and ≥ 32) 
 Gender (male and female) 
 Iron chelation therapy (yes and no) 
 Baseline LIC (< 3, 3-5, ≥ 5) 
 Baseline EPO (< 200 and ≥ 200), (< 200, 200-500, and ≥ 500), (< 500, and ≥ 500)   
 Splenectomy (yes and no) 
5.8. 
Safety Analysis 
The safety endpoints will be summarized by dose cohort using the Safety Population.  The 
safety endpoints include the incidence of dose-limiting toxicities (DLTs), treatment emergent 
adverse events, changes in laboratory tests, vital signs and ECG’s. 
Adverse events will be coded using MedDRA Version 15.1 and above. Severity of AEs will be 
coded using National Cancer Institute Common Toxicity Criteria for Adverse Events version 
4.0 (NCI-CTCAE v4.0). 
5.8.1. 
Dose Limiting Toxicities  
A Dose-Limiting Toxicity (DLT) is defined as any of the following toxicities, at any dose 
level occurring within 28 days of the first administered dose:  
 Treatment related serious adverse event (SAE) of Grade ≥ 3  
 Treatment related non-hematologic adverse event (AE) of Grade ≥ 3  
 Treatment related hematologic AE of Grade ≥ 4  
Incidence rates of DLTs will be presented by dose cohort. Details of DLTs will be presented 
in listings. 
5.8.2. 
Adverse Events 
All AEs and SAEs occurring after the Cycle 1 Day 1 dose through 56 days after the last study 
drug administration are to be reported and documented on the AE CRF.  All AEs collected in 
this study are treatment emergent adverse events (TEAE).  A drug-related TEAE is defined as 
any TEAE related to the study medication as assessed by the investigator or with missing 
assessment of the causal relationship. 
The following summaries will be presented by dose cohort and by low/high dose for all dosed 
patients as well as for NTD and TD patients separately: 


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 Number and percentage of patients reporting each AE, categorized by System 
Organ Class (SOC) and Preferred Terms (PT) 
 Number and percentage of patients reporting each AE experienced by ≥ 5% of 
patients in all patients by PT 
 Number and percentage of patients reporting SAE, categorized by System Organ 
Class (SOC) and Preferred Terms (PT) 
 Number and percentage of patients reporting Grade ≥ 3 AE, categorized by 
System Organ Class (SOC) and Preferred Terms (PT) 
 Number and percentage of patients reporting related AE, categorized by System 
Organ Class (SOC) and Preferred Terms (PT) 
 Number and percentage of patients reporting AE leading to drug withdrawal, 
categorized by System Organ Class (SOC) and Preferred Terms (PT) 
 Number and percentage of patients reporting each AE categorized by SOC, 
preferred term, and maximum severity grade group 
Note that counting will be by subject, not event, and subjects are only counted once within 
each SOC or preferred term.  If a subject experiences the same AE at more than one severity, 
or with more than one relationship to study drug, the most severe rating or the stronger causal 
relationship to study drug will be given precedence.  Any missing severity, causality, or 
outcome will not be imputed and classed as unknown. 
All AEs will be listed.  The following listings will also be provided: 1) patients with SAEs; 2) 
patients with Grade ≥ 3 AEs; and 3) patients with AEs leading to discontinuation 
5.8.3. 
Laboratory Evaluations 
Results from the following laboratory parameters, recorded at their respective time points 
will be summarised by dose cohort and time point:  
Haematology: (Screening, Days 1, 8 and 15 of each cycle, End of Treatment, Post Treatment 
Follow Up and End of Study). 
RBC, WBC with differential, hemoglobin, hematocrit, haptoglobin (optional), reticulocyte 
count, platelet count, MCV, MCH, MCHC, RDW, and nRBCs   
Chemistry: (Screening, Days 1 and 15 of Cycle 1, and Days 1 of each subsequent cycle, End 
of Treatment and End of Study). 
Sodium, potassium, chloride, carbon dioxide/bicarbonate (optional), AST, ALT, lactate 
dehydrogenase (LDH), total bilirubin, indirect bilirubin, alkaline phosphatase, blood urea 
nitrogen (BUN)/urea, creatinine, GGT, calcium, phosphorus, glucose, amylase, lipase, total 
protein, albumin, and uric acid 
Urine: (Day 1 of Cycles 1, 2 and 4, End of Treatment and End of Study). 
PH, specific gravity, protein, glucose, ketones, blood, leukocyte esterase, and nitrite, with 
microscopic examination if indicated; Microalbumin and creatinine  
Urine Chemistry: (Day 1 of Cycles 1, 2 and 4, End of Treatment and End of Study).   
Microalbumin and creatinine will be performed by the central lab.  
Categorical and numeric variables will be presented separately. 


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Actual values and changes in haematology and biochemistry laboratory values from baseline 
will be summarised by dose cohort and by time point. 
Shift tables for the haematology and biochemistry laboratory parameters comparing values 
above, within and below the normal reference range at baseline to the end of treatment visit 
will be presented using standard reference ranges.  
Actual urine laboratory values will be summarised by dose cohort. 
Peripheral blood smears are taken at Screening, Day 1 of Cycles 1, 2, and 5 and at the end of 
treatment visit.  These data will be listed only. 
All laboratory values will be listed for all patients. 
5.8.4. 
Vital Signs 
Vital signs parameters include weight (kg), heart rate, systolic and diastolic blood pressure, 
respiratory rate, and temperature (◦C).  Vital signs are recorded at Screening and on Days 1, 8 
and 15 of each cycle and EOT. For each parameter at each time point, the change from baseline 
to post baseline will be summarised by dose cohort.  The baseline assessment is defined as the 
last measurement on or before Cycle 1 Day 1.  
Plots of vital sign parameters for both mean observed and mean change from baseline will be 
presented by dose cohort for each parameter. 
Vital signs will also be listed for all patients. 
5.8.5. 
ECG Results 
12 lead ECG results are recorded at Screening, Cycle 2 Day 1, and at the end of treatment 
visit. The quantitative ECG assessments (Ventricular rate, QRS width, PR interval, and QTc 
interval) will be summarised at each time point by dose cohort. 
ECG overall interpretation (normal, abnormal not clinically significant and abnormal 
clinically significant) will be presented for actual values and changes from baseline 
(Screening observation) to each post baseline visit [expressed as Improvement, No Change, 
and Deterioration] by dose cohort. 
Note: 
 Improvement = Abnormal Clinically Significant (CS) to Abnormal Not Clinically 
Significant (NCS)/Normal, Abnormal NCS to Normal  
 Deterioration = Normal to Abnormal NCS/CS, Abnormal NCS to Abnormal CS  
 No change = Normal to Normal, Abnormal NCS to Abnormal NCS, Abnormal CS 
to Abnormal CS 
If either result is missing for any subject, then an ‘Unknown’ category will be presented. 
ECG results will be listed for all patients. 
5.8.6. 
Physical Examination 
A physical examination is conducted at Screening and on Day 1 of each cycle and at the end 
of treatment and end of study visits.  Physical exam details will be listed only. 


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5.9. 
Pharmacokinetics Analysis 
5.9.1. 
Pharmacokinetic Sampling Schedule  
Blood samples for determination of ACE-536 serum concentrations are collected from all 
subjects at the following schedule: 
Treatment/Follow Up 
Visit 
Study Day 
PK Sample 
Dose 1 
Cycle 1 Day 1 
1 
X 
 
Cycle 1 Day 8 
8 
X 
 
Cycle 1 Day 11 
11 
X 
 
Cycle 1 Day 15 
15 
X 
Dose 2 
Cycle 2 Day 1 
22 
X 
 
Cycle 2 Day 8 
29 
X 
Dose 4 
Cycle 4 Day 1 
64 
X 
 
Cycle 4 Day 8 
71 
X 
 
Cycle 4 Day 15 
78 
X 
Dose 5 (Final Dose) 
Cycle 5 Day 1 
85 
X 
 
Cycle 5 Day 8 
92 
X 
 
Cycle 5 Day 15 
99 
X 
Follow up Period 
End of Treatment 
113 
X 
 
Post-Treatment Follow Up 
141 
X 
 
End of Study 
169 
X 
Acceptable time windows for pharmacokinetic blood draws are ± 1 day in Cycle 1, ± 2 days 
in Cycles 2 to 5, and ± 7 days in the follow-up period. 
5.9.2. 
Data Handling  
Concentrations that are below the limit of quantitation (BLQ) prior to the first dose will be 
assigned a numerical value of zero.  Post-treatment concentrations that are BLQ will be 
treated as missing.    
Concentrations assigned a value of missing will be omitted from the descriptive statistics.  A 
concentration value of zero will be excluded from the computation of the geometric mean 
(geometric CV%).  If any subjects are found to be noncompliant with respect to dosing, have 
incomplete data, or encounter other circumstances that would affect the evaluation of 
pharmacokinetics, a decision will be made on a case-by-case basis as to their inclusion in the 
pharmaockinetic analysis.  Data excluded from pharmacokinetic analysis will be included in 
the data listings, but not in the summaries. 
In tables and listings for the derived pharmacokinetic data, there should be four decimal 
places for numerical values below 1, three decimal places for numeric values below 10 but 
above 1, and two decimal places for numeric values above 10.  However, the listings of raw 
data should not have more decimal places than the actual data. 


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5.9.3. 
 Noncompartmental Pharmacokinetic Analysis 
Pharmacokinetic parameters of ACE-536 will be derived using noncompartmental analysis 
method with the software program Phoenix WinNonlin (version 6.2 or higher).  The 
ACE-536 serum concentrations after the first dose (Cycle 1 Day 1 to Cycle 2 Day 1) will be 
used to estimate the drug serum exposure parameters within the first treatment cycle, and the 
ACE-536 serum concentrations after the final dose (Cycle 5 Day 1 to end of study), if 
sufficient, will be used to estimate the terminal elimination half-life in serum.  Actual 
sampling and dosing times will be used in this analysis.  The following noncompartmental 
pharmacokinetic parameters will be estimated: 
Tmax 
Time to maximum observed serum concentration, obtained directly from the 
observed concentration-time data (for the first dose only) 
Cmax 
Maximum observed serum concentration, obtained directly from the 
observed concentration-time data (for the first dose only) 
AUC21d 
Area under the serum concentration-time curve from Day 1 to Day 21, 
calculated  by the linear trapezoidal method (for the first dose only) 
λz 
Apparent terminal rate constant, calculated by linear regression of the 
terminal portion of the log-concentration-time curve in serum.  Visual 
assessment may be used to identify the terminal linear phase of the log 
concentration-time profile.  A minimum of 3 data points will be used for the 
calculation.  The λz will not be estimated if the terminal phase of the log-
concentration-time profile does not exhibit a linear decline phase, or if the 
regression coefficient (Rsq) is less than 0.8 (after the final dose) 
t1/2 
Terminal elimination half-life in serum, calculated as [(ln2)/λz].  The 
terminal elimination half-life will only be calculated when a reliable estimate 
for λz can be obtained (after the final dose) 
The following pharmacokinetic parameters will be calculated for diagnostic purposes and 
listed, but they will not be summarized: 
λz lower: lower limit of time (days) included in the calculation of λz 
λz upper: upper limit of time (days) included in the calculation of λz 
λz N: number of data points used in the calculation of λz 
Rsq: regression coefficient for calculation of λz 
Additional noncompartmental pharmacokinetic parameters may be determined when 
appropriate. 
5.9.4. 
Compartmental Pharmacokinetic Analysis 
Pharmacokinetic parameters of ACE-536 will also be derived using a one-compartment 
model with first order absorption and elimination (Phoenix WinNonlin).  All ACE-536 serum 
concentration values obtained from Cycle 1 Day 1 to the end of study will be included in this 
analysis.  Actual sampling and dosing times will be used in this analysis.   
The model will be parameterized in terms of the absorption rate constant (k01), apparent 
clearance (CL/F), and apparent volume of distribution (V/F).  Other parameters will be 
derived from the established model.  The main compartmental pharmacokinetic parameters to 
be estimated are outlined as following: 


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k01 
Absorption rate constant 
CL/F 
Apparent clearance from the central compartment 
V/F 
Apparent volume of distribution of the central compartment 
Tmax 
Time to maximum serum concentration, calculated as ln(k01/k10)/(k01-k10), 
where k10 = (CL/F)/(V/F) 
Cmax 
Maximum serum concentration for the starting dose, predicted at tmax  
AUC 
Area under the serum concentration-time from time zero to infinity for the 
starting dose, calculated as (Starting Dose)/(CL/F).  
t1/2 
Elimination half-life, calculated as (ln2)/k10 
Additional compartmental pharmacokinetic parameters may be determined when appropriate. 
5.9.5. 
Statistical Analysis  
All ACE-536 serum concentrations will be listed by dose group, subject, and scheduled time 
(visit and study day).  The ACE-536 serum concentrations will be summarized by dose group 
and scheduled time, including N (number of observations), arithmetic mean, arithmetic 
standard deviation (SD), arithmetic coefficient of variation (CV%), geometric mean, 
geometric CV%, minimum, median, and maximum.  Mean (SD) serum concentration-time 
profiles will be presented on both linear and semilogarithmic scales by dose group for the 
first 4 weeks.  Individual concentration plots will be provided by presenting the observed 
concentration data along with the one-compartment model-predicted concentration-time 
profile on the linear scale using actual sampling times and multiple dose data. 
Pharmacokinetic parameters will be listed by dose group and subject, and they will be 
summarized descriptively by dose group (N, mean, SD, minimum, median, maximum, CV%, 
geometric mean, and geometric CV%).  Dose proportionality may be assessed using the 
exposure data (e.g., Cmax, AUC) after the first dose.  The relationship between ACE-536 
exposure and response (i.e., safety, efficacy, and biomarkers) may be explored, if appropriate.   
5.10. 
Interim Analysis 
There are no planned interim analyses.  However, safety and erythroid response data will be 
reviewed periodically throughout the study. 
5.11. 
Protocol Deviations 
Protocol deviations will be listed for the following categories: 
 Not meeting inclusion/Exclusion criteria 
 Withdraw criteria met, but subject not discontinued: we not have any patients who 
fall in this category; will be noted as such in CSR 
 Failure to perform key procedures  
 Study treatment deviation 
 Prohibited concomitant medication 
 GCP related deviation 


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5.12. 
Handling of Missing Data  
As a general principle, no imputation of missing data for other variables will be done. 
Exceptions are the start and stop dates of AEs and concomitant medication with the rules 
listed below.  The imputed dates will be used to allocate the medication as prior or 
concomitant medications and to determine whether an AE is/is not treatment emergent. 
Listings of the AEs and concomitant medications will present the actual partial dates; 
imputed dates will not be shown. 
In addition, partial disease diagnosis dates are imputed to calculate time since disease 
diagnosis.  The listing will present the actual partial dates.   
5.12.1. 
Missing Dates for Adverse Event 
a) Imputing partial AE start dates: If the year is unknown, the date will not be imputed 
and will be assigned a missing value. 
b) If the month is unknown, then: 
1) If the year matches the first dose date, then impute the month and day of the 
first dose date. 
2) Otherwise, assign January. 
c) If the day is unknown, then: 
1) If the month and year match the first AE stop month and year and AE stop day 
is not missing, then impute the start day as the stop day. 
2) Otherwise impute start day using the last day of the start month. 
Imputing partial AE stop dates: 
a) If the year is unknown, the date will not be imputed and will be assigned a missing 
value. 
b) If the month is unknown, then assign December. 
c) If the day is unknown, then assign the last day of the month. 
5.12.2. 
Missing Dates for Concomitant Medication  
If start date is missing or partial: 
a) if month is missing, use January  
b) if day is missing, use the first day of the month under consideration 
c) if year is missing, use year of the informed consent date  
d) if entire date is missing, use informed consent date  
If stop date is missing or partial: 
a) if month is missing, use December  
b) if day is missing, use the last day of the month under consideration 
c) if year or the entire date is missing, set to December 31st, 2099 
If the imputed start date is after the stop date, then the imputed start date will be one day prior 
to the stop date. 


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5.12.3. 
Missing Dates for Disease Diagnosis Date  
For disease diagnosis dates, the imputation rules are  
a) if day is missing, use 15th of the month 
b) if both day and month are missing, impute as January 1st 
c) if month is missing, impute as January 
d) if year is missing, set to missing 
5.13. 
Changes in Conduct or Planned Analyses from the Protocol 
Major changes between SAP and the planned analysis in Protocol Amendment 4 are 
described below: 
Changes 
Rationale 
Definition of 
baseline 
hemoglobin 
Protocol definition: Baseline hemoglobin will be the average of at least two 
measures (not influenced by transfusion within 7 days of measurement); one 
measure performed within one day prior to Cycle 1 Day 1 and the other 
performed 7-28 days prior to Cycle 1 Day 1.   
This definition is revised in this SAP to reflect the derivation rule 
implemented for this study. 
Remove Efficacy 
Evaluable (EE) 
population 
EE population is very similar to ITT population. ITT provides more 
conservative estimate of treatment effect.  
6. 
REFERENCES 
National Cancer Institute Common Toxicity Criteria for Adverse Events, version 4.0 
(NCI-CTCAE v4.0) 
7. 
APPENDICES 
7.1. 
FACT-An Scoring Algorithm 
The Fact-An questionnaire contains 47 questions which are divided into the following sub 
scales: 
 Physical Well-Being (PWB) (7 questions: Item Score range 0 - 28) 
 Social/Family Well-Being (SWB) (7 questions: Item Score range 0 - 28) 
 Emotional Well-Being (EWB) (6 questions: Item Score range 0 - 24) 
 Functional Well-Being (FWB) (7 questions: Item Score range 0 - 28)  
 Anemia Subscale (AnS) (20 questions : Item Score range 0 - 80) 
 Fatigue Subscale (FACIT-F) (13 questions : Item Score range 0 – 52) 
 Fatigue Experience (FE) (5 questions : Item Score range 0 – 20) 
 Fatigue Impact (FI) (8 questions : Item Score range 0 – 32) 


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Subjects give individual responses to each question on a scale of 0 to 4 (0=Not at all; 1=A 
little bit; 2=Somewhat; 3=Quite a bit; 4=Very much).  Item scores in the PWB and EWB sub 
scales will be derived by subtracting the response value from 4.  Similarly, all Item Scores in 
the AnS subscale except Item Codes An5, An7, BL4 and An133 will be derived by 
subtracting the response value from 4.  Thus, a higher Item Scores indicates a better quality 
of life.   
Subscale totals will be derived as follows: 
Sum of Item Scores x Number of Items in Subscale 
Number of Items Answered 
For example, if 6 questions are answered in the Physical Well-Being subscale and the Item 
scores sum to a total score 18 the subscale score will be (18 x 7)/6 = 21. 
If 50% or more of responses in any subscale are missing the subscale total will be set to missing. 
The following total scores will also be derived: 
FACT-An Total Outcome Index (TOI) derived as: PWB + FWB + AnS (Score range 0 – 136) 
FACT-G Total Score (FACT-G) derived as: PWB + SWB + EWB + FWB (Score range 
0 – 108) 
FACT-An Total Score (FACT-An) derived as: PWB + SWB + EWB + FWB + AnS 
(Score range 0 – 188) 
If 20% or more of the responses that contribute to the FACT-G score are missing the FACT-G 
score will be set to missing (ie at least 22 of the 27 items contributing to FACT-G must be 
present). Furthermore, FACT-An TOI, FACT-G and FACT-An scores should only be 
calculated if all component subscales have valid scores.  If any subscale total is missing the 
respective total scores to which the subscale contributes will also be set to missing.  A higher 
FACT total score indicates a better quality of life. 
7.2. 
SF-36 Scoring Algorithm 
The 36 individual items scores will be used to compute the 8 domain scores (Physical 
Functioning, Role-Physical, Bodily Pain, General Health, Vitality, Social Functioning, 
Role-Emotional, and Mental Health).  The 8 domains scores will be transformed to a scale 
from 0 to 100.  The transformed scores will be used in the analysis. The Physical Component 
Scores (PCS) and Mental Component Scores (MCS) will be computed from the transformed 
domain scores. 
The scoring of the domain and component scores will follow the following steps: 
 Reverse code 10 of the items (Items 1, 6, 7, 8, 9a, 9d, 9e, 9h, 11b and 11d).  Algorithm 
for reverse coding for those 10 items is as follows: 
1) If (Item 1 = 1 or 4 or 5) then Item 1 = 6 – Item1, 
Else if Item 1 = 2 then Item 1 = 4.4, 
Else if Item 1 = 3 then Item 1 = 3.4 
2) Item 6 = 6 – Item 6 
3) If (Item 7 = 1 or 6) then Item 7 = 7 – Item 7, 
Else if Item 7 = 2 then Item 7 = 5.4, 


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Else if Item 7 = 3 then Item 7 = 4.2, 
Else if Item 7 = 4 then Item 7 = 3.1, 
Else if Item 7 = 5 then Item 7 = 2.2 
4) Reverse scoring of Item 8 depends on whether Item 7 is answered. 
If Item 7 is answered, then recode Item 8 as follows:  
If precoded Item 7 = 1 and Item 8 = 1 then Item 8 = 6, 
Else Item 8 = 6 – Item 8. 
If Item 7 is not answered, then recode Item 8 as follows: 
If Item 8 = 1 then Item 8 = 6, 
Else if Item 8 = 2 then Item 8 = 4.75, 
Else if Item 8 = 3 then Item 8 = 3.5, 
Else if Item 8 = 4 then Item 8 = 2.25, 
Else if Item 8 = 5 then Item 8 = 1 
5) Item 9a = 6 – Item 9a 
6) Item 9d = 6 – Item 9d 
7) Item 9e = 6 – Item 9e 
8) Item 9h = 6 – Item 9h 
9) Item 11b = 6 – Item 11b 
10) Item 11d = 6 – Item 11d 
 Compute raw scores of each domain by summing up the items in each domain.  A domain 
score will be computed only if at least half of the items in the domain are non-missing.  If 
at least half of the items in the domain are non-missing, missing score in the same domain 
will be replaced by the average value of the non-missing scores.  Recoded scores will be 
used in the summation and the imputation. The raw domain scores will then be 
transformed into a scale of 0 to 100 based on their lowest and highest value, using the 
following formula: 
 
 


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Transformed Score = ((Actual – Lowest)/(Highest – Lowest)) x 100 
SF-36 Domain Score Items 
 
 
 
 
Domain 
Items 
Lowest 
Highest 
Physical Functioning 
(PF) 
Items 3a, 3b, 3c, 3d, 3e, 3f, 
3g, 3h, 3i, 3j 
10 
30 
Role-Physical (RP) 
Items 4a, 4b, 4c, 4d 
4 
20 
Bodily Pain (BP) 
Items 7 and 8 
2 
12 
General Health (GH) 
Items 1, 11a, 11b, 11c, 11d 
5 
25 
Vitality (VT) 
Items 9a, 9e, 9g, 9i 
4 
20 
Social Functioning 
(SF) 
Items 6 and 10 
2 
10 
Role-Emotional (RE) 
Items 5a, 5b, 5c 
3 
15 
Mental Health (MH) 
Items 9b, 9c, 9d, 9f, 9h 
5 
25 


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 Transformed score will be standardized to z scores based on the following formulas: 
PF_Z = (PF – 83.29094) / 23.75883 
RP_Z = (RP – 82.50964) / 25.52028 
BP_Z = (BP – 71.32527) / 23.66224 
GH_Z = (GH – 70.84570) / 20.97821 
VT_Z = (VT – 58.31411) / 20.01923 
SF_Z = (SF – 84.30250) / 22.91921 
RE_Z = (RE – 87.39733) / 21.43778 
MH_Z = (MH – 74.98685) / 17.75604 
 Transform z scores to T scores based on the following formulas: 
PF_T=50+PF_Z*10 
RP_T=50+RP_Z*10 
BP_T=50+BP_Z*10 
GH_T=50+GH_Z*10 
VT_T=50+VT_Z*10 
SF_T=50+SF_Z*10 
RE_T=50+RE_Z*10 
MH_T=50+MH_Z *10 
 The aggregate physical (AGG_PHYS) and mental (AGG_MENT) component scores are 
computed as: 
AGG_PHYS = (PF_Z * .42402) + (RP_Z * .35119) + (BP_Z * .31754) +  
(GH_Z * .24954) + (VT_Z * .02877) + (SF_Z * -.00753) + 
(RE_Z * -.19206) + (MH_Z * -.22069) 
AGG_MENT = (PF_Z * -.22999) + (RP_Z * -.12329) + (BP_Z * -.09731) +  
(GH_Z * -.01571) + (VT_Z * .23534) + (SF_Z * .26876) + 
(RE_Z * .43407) + (MH_Z * .48581) 
 Physical Component Scores (PCS) and Mental Component Scores (MCS) are computed 
as: 
PCS = 50 + (AGG_PHYS * 10) 
MCS = 50 + (AGG_MENT * 10) 
 
 


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8. 
LIST OF ABBREVIATIONS 
Abbreviation 
Definition 
AE 
Adverse Event 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase 
AUC 
Area under the concentration-time curve 
BMD 
Bone mineral density 
BMP 
Bone morphogenetic protein 
BSAP 
Bone specific alkaline phosphatase 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CI 
Confidence interval 
Cmax 
Maximum concentration 
CREAT 
Creatinine 
CRF 
Case report form 
CRO 
Contract research organization 
CTX 
C-telopeptide of type I collagen 
DLT 
Dose-limiting toxicity 
ECHO 
Echocardiogram 
ECG 
Electrocardiogram 
EE 
Efficacy Evaluable 
EOS 
End of Study 
EPO 
Erythropoietin 
EOT 
End of Treatment 
FACT-An 
Functional Assessment of Cancer Therapy-Anemia Scale 
HbA 
Adult hemoglobin 
HbF 
Fetal hemoglobin 
HTB 
High Transfusion Burden 
ICF 
Informed consent form 
IEC 
Independent ethics committee 
ITT 
Intent-to-Treat 
IB 
Investigator’s brochure 
LDH 
Lactate dehydrogenase 
LTB 
Low Transfusion Burden 
MedDRA 
Medical Dictionary for Regulatory Activities 


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Abbreviation 
Definition 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
MRI 
Magnetic resonance imaging 
MUGA 
Multi gated acquisition scan 
NCI-CTCAE 
National Cancer Institute-Common terminology criteria for adverse events 
nRBCs 
Nucleated red blood cells 
NTBI 
Non-transferrin bound iron  
PD 
Pharmacodynamic 
PK 
Pharmacokinetic 
QoL 
Quality of life 
RBC 
Red blood cell 
RDW 
Red blood cell distribution width 
RNA 
Ribonucleic acid 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SC 
Subcutaneous 
SD 
Standard deviation 
SRT 
Safety review team 
SUSAR 
Suspected unexpected serious adverse reaction 
T1/2 
Elimination half-life 
TGF-β 
Transforming growth factor beta 
TIBC 
Total iron binding capacity 
Tmax 
Time to maximum concentration 
ULN 
Upper limit of normal 
WBC 
White blood cell 


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9. 
SCHEDULE OF EVENTS 
 


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Screen 
Treatment Period 
Follow up period 
Cycle 1 
Cycle 2 
Cycle 3 
Cycle 4 
Cycle 5 
EOT12 
EOS13 
C1D
12 
C1D8 
C1D1
1 
C1D1
5 
C2D12,
15 
C2D8 
C2D15 
C3D12,15 
C3D8 
C3D15 
C4D12,15 
C4D8 
C4D1
5 
C5D12,
15 
C5D8 
C5D15 
Day 113 
(± 7d) 
Day 
141 
(± 7d) 
Day -28 to 
-1 
Day 
1 
Day 
8 
(± 1d) 
Day 
11 
(± 1d) 
Day 
15 
(± 1d) 
Day14 
22 
(± 2d) 
Day 
29 
(± 2d) 
Day 
36 
(± 2d) 
Day14 
43 
(± 2d) 
Day 
50 
(± 2d) 
Day 
57 
(± 2d) 
Day14 
64 
(± 2d) 
Day 
71 
(± 2d) 
Day 
78 
(± 2d) 
Day14 
85 
(± 2d) 
Day 
92 
(± 2d) 
Day 
99 
(± 2d) 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion  
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
QoL Questionnaires19 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X20 
X 
Physical examination 
X 
X23 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X23 
X23 
Vital signs1  
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
ECG (12 lead) 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
MRI (Liver Iron Content)17 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
MRI for EMH masses and 
spleen24 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
DXA for BMD of total 
body, lumbar spine, and 
total hip24 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Leg Ulcer Assessment25 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Abdominal Ultrasound21 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
ECHO, MUGA or cardiac 
MRI 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6MWT24 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Serum iron studies4 
X 
X 
X 
 
X 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Serum folate and B12 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Erythropoietin levels  
X 
X 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
X 
Hematology5 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Peripheral blood smear 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
Serum chemistry6 
X 
X 
 
 
X 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Urinalysis and Urine 
Chemistry7 
X 
X 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
X 
Anti-drug antibody16  
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
X16 
PK collection 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
PD Biomarkers8 
 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Hemoglobin electrophoresis 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
Globin mRNA sample 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
Bone Biomarkers9 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
Pregnancy test/menstrual 
history10 
X 
 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X22 
Evaluate transfusion 
frequency/volume11 
X 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Concomitant medications 
and AEs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Administer ACE-53618  
 
X 
 
 
 
X14 
 
 
X14 
 
 
X14 
 
 
X14 
 
 
 
 


A536-04 
 
Statistical Analysis Plan 
Confidential and Proprietary 
31 
Acceleron Pharma Inc. 
1  
Vital signs: Weight, heart rate, systolic and diastolic blood pressure, respiration rate, and temperature (measured in degrees Celsius). Height is measured only at Screening.  If at 
any visit the systolic blood pressure is ≥ 150 mmHg, the diastolic blood pressure is ≥ 95 mmHg, and/or the absolute increase in either measure from baseline is ≥ 20 mmHg, 
perform one repeat of the blood pressure assessment after a minimum of 15 minutes.   
2  Study procedures must be done prior to administration of study drug.    
3  Screening MRI for Liver Iron Content, MRI of the chest and abdomen for EMH masses and spleen (expansion cohort only), BMD by DXA (expansion cohort only), abdominal 
ultrasound of the spleen (if no prior splenectomy; dose escalation cohorts only) and ECHO, MUGA or cardiac MRI can be performed up to 56 days prior to C1D1.  If performed as part 
of standard of care, it does not need to be repeated. 
4  Iron Studies: Serum iron, TIBC, transferrin, soluble transferrin receptor, ferritin, and NTBI. 
5  
Hematology: RBC, WBC with differential, hemoglobin, hematocrit, haptoglobin (optional), reticulocyte count, platelet count, MCV, MCH, MCHC, RDW, and nRBCs.  On 
dosing days, hematology values are to be drawn and resulted (up to 1 day) prior to C1D1.  Historical hemoglobin values will be collected for 12 weeks prior to C1D1.  Historical 
transfusion history will be collected for 12 months prior to Cycle 1 Day 1.  Baseline hemoglobin will be the average of two measurements; one measure performed within one 
day prior to Cycle 1 Day 1 and the other performed during the screening period (Day -28 to Day -1).  Note: For any RBC transfusions received during the study, collect 
hemoglobin value just prior to transfusion.  
 6  Chemistry: Sodium, potassium, chloride, carbon dioxide/bicarbonate (optional), AST, ALT, lactate dehydrogenase (LDH), total bilirubin, indirect bilirubin, alkaline 
phosphatase, blood urea nitrogen (BUN)/urea, creatinine, GGT, calcium, phosphorus, glucose, amylase, lipase, total protein, albumin, and uric acid.  
7  Urinalysis by dipstick analysis: pH, specific gravity, protein, glucose, ketones, blood, leukocyte esterase, and nitrite, with microscopic examination if indicated.  Microalbumin and 
creatinine will be performed by the central lab. 
8   PD Biomarkers: Hepcidin, GDF15 and others to be determined. 
9   Bone Biomarkers: BSAP and CTX. 
10    Pregnancy test (urine or serum) and menstrual history is required prior to C1D1 for female patients of child bearing potential only. 
11  Transfusion history will be collected for 12 months prior to C1D1. 
12  End of Treatment (EOT): Should be performed 28 days (± 7 days) after the last dose of ACE-536.  Patients who discontinue treatment early should complete the end of 
treatment visit at the time of discontinuation and complete the Day 141 follow up visit 28 days (± 7 days) after the EOT visit. 
13  End of Study (EOS): Should be performed 28 days (± 7 days) after the Day 113/EOT visit (56 days after the last dose of ACE-536).  
14  Day 85 ± 2 days is the last possible study day that ACE-536 may be administered, regardless of the cycle. 
15  If a dose delay is required per the dose modification rules the patient will not be dosed. The patient will return weekly to assess hematology results and adverse events until the patient is 
eligible to administer the next dose of ACE-536. 
16  
 If the patient has a positive ADA result at their last assessment, the patient may be asked to return approximately every three months for additional testing until a negative result is 
obtained or the result is considered stabilized. 
17   MRI for liver iron content will be performed at selected sites. 
18 For the first dose of ACE-536, dosing should occur after a minimum of 7 days post-transfusion; subsequent doses should not be given within 24 hours of transfusion or planned 
transfusion. 
19  Administration of quality of life questionnaires, including but not limited to FACT-An and SF-36 is required for patients in the expansion cohort only. 
20  QoL questionnaires including but not limited to FACT-An and SF-36 should be completed at the EOT visit only for expansion cohort patients who discontinue treatment early. 
21  Abdominal ultrasound of the spleen is required for patients with no prior splenectomy in the dose escalation cohorts only.  Patients in these cohorts with clinical signs of a change in 
spleen size or abnormality should have an abdominal ultrasound as needed throughout study. 
22  Pregnancy test is not required at Day 141/EOS. 
23  Physical exam should include an optional evaluation of gonadal size in males at C1D1, EOT, and EOS visits only. 
24  MRI of the chest and abdomen for EMH masses and spleen, DXA for BMD and 6MWT for NTD patients will be performed in the expansion cohort patients only, at selected sites. 
25  Patients with leg ulcers should have regular assessment of the leg ulcer(s) throughout the study.  Photographs of the leg ulcer(s) should be obtained to document any changes in leg 
ulcer(s) size.