Clinical trial NCT02268409
Conditions: β-Thalassemia
Sponsor: Acceleron Pharma, Inc., a wholly-owned subsidiary of Merck & Co., Inc., Rahway, NJ USA
Start date: 2014-10-20; Completion date: 2020-06-18
Enrollment: 51 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Study A536-06 (MK-6143-004) is an open-label extension study for participants previously enrolled in study A536-04 (NCT01749540), to evaluate the long-term safety and tolerability of luspatercept in adult participants with beta-thalassemia.

Detailed description:

Arms: arm 1: Participants receive luspatercept 0.6, 0.8, 1.0, or 1.25 mg/kg administered by subcutaneous injection on Day 1 of each 21-day cycle for up to 87 cycles (up to approximately 5 years). Participants will receive the highest tolerated dose level of luspatercept that they were assigned in the base study unless a dose modification was required.
Interventions: intervention 1: luspatercept
Intervention descriptions: intervention 1: subcutaneous injection

Protocol documents:
Prot_000.pdf: 
Official Protocol Title: 
NCT number: 
Document Date: 
An Open-Label Extension Study to Evaluate the Long-Term 
Effects of ACE-536 in Patients with β-Thalassemia Previously 
Enrolled in Study A536-04
29-Mar-2019
NCT02268409


Protocol A536-06  
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
          CONFIDENTIAL 
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CLINICAL STUDY PROTOCOL 
An Open-Label Extension Study to Evaluate the Long-Term Effects of ACE-536 
in Patients with β-Thalassemia Previously Enrolled in Study A536-04 
 
    INVESTIGATIONAL PRODUCT:  
 
Luspatercept (ACE-536) 
 
    PROTOCOL NUMBER:  
 
 
 
 
A536-06 
 
EUDRACT NUMBER: 
2014-001281-94 
 
 
 
SPONSOR: 
Acceleron Pharma Inc. 
128 Sidney Street 
Cambridge, MA 02139 USA 
 
Tel: 
 
Fax: 
 
 
   MEDICAL MONITOR:  
 
 
 
 
 
 
 
 
 
 
 
Vice President, Medical Research 
 
 
 
 
ORIGINAL PROTOCOL DATE: 
 
 
14-Apr-2014 
 
AMENDMENT 01 DATE: 
 
 
 
 
08-Dec-2014 
 
AMENDMENT 02 DATE:                             03-Jun-2015   
 
AMENDMENT 03 DATE:                             23-May-2016   
 
AMENDMENT 04 DATE:                             16-Aug-2016   
 
AMENDMENT 05 DATE:  
 
 
 
 
12-Sept-2017 
 
AMENDMENT 06 DATE: 
 
 
 
 
29-Mar-2019 
 
Confidentiality Statement 
The 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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COORDINATING PRINCIPAL INVESTIGATOR SIGNATURE PAGE 
 
 
 
 
 
 
Signature: 
 
 
 
Date: 
 
 
 
Name (print): 
Prof. Antonio Piga 
 
 
DD/MMM/YYYY 
 
 
 
 
 
 
Institution Name and Address: 
 
 
 
 
University of Torino 
 
 
Department of Clinical and Biological Sciences 
 
 
Regione Gonzole 10 – 10043 Orbassano  
 
 
Turin, Italy 
By my signature, I agree the protocol has been written to comply with International Conference 
of Harmonization (ICH) Good Clinical Practices (GCP) guidelines and agree to offer guidance 
throughout the study as needed. 
I agree to 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, ICH 
Guidelines, GCP, the Declaration of Helsinki, and local ethical and legal requirements. 
 
 
 
 
 
 
 
 


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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 FDA 
regulations, ICH Guidelines, 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 
Direct Line:  
 
Mobile: 
 
Fax:  
 
 
Chiltern Medical Advisor 
 
 
Chiltern 
Via M. Nizzoli, 6 
Milano 20147 Italia 
Direct Line: 
 
Mobile: 
 
Fax: 
 
 
Pharmacovigilance  
Chiltern 
 
 
 
 
 
 
 
PPD
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PPD
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Protocol A536-06  
 
 
 
 
 
 
 
 
 
 
 
 
 
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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  
Title of Study: An Open-Label Extension Study to Evaluate the Long-Term Effects of 
ACE-536 in Patients with β-Thalassemia Previously Enrolled in Study A536-04 
Study Centers: up to 15 
Phase of Development: 2 
Objectives: 
Primary:  
 To evaluate the long-term safety and tolerability of ACE-536 in patients with β-thalassemia 
who were previously enrolled in study A536-04. 
Secondary: 
 To evaluate erythroid response defined as the proportion of patients with: 
o Mean hemoglobin increase ≥ 1.5 g/dL over a continuous 12-week interval compared 
to baseline in non-transfusion dependent (NTD) patients, not influenced by red 
blood cell (RBC) transfusion, OR 
o Reduction in RBC transfusion burden by ≥ 50% over a continuous 12-week interval 
compared to the 12 weeks prior to the start of treatment in transfusion dependent 
(TD) patients. 
 To evaluate erythroid response defined as proportion of patients with: 
o Mean hemoglobin increase ≥ 1.5 g/dL over a continuous 8-week interval compared 
to baseline in NTD patients, not influenced by RBC transfusion, OR 
o Reduction in RBC transfusion burden by ≥ 20% over a continuous 8-week interval 
compared to the 8 weeks prior to the start of treatment in TD patients. 
 To evaluate the time to erythroid response and duration of erythroid response. 
 To evaluate the mean change from baseline over an 8- or 12-week period in hemoglobin 
level in NTD patients not influenced by RBC transfusion 
 To evaluate the mean % change from baseline in transfusion burden over an 8- or 12-week 
period in TD patients. 
 To evaluate the mean change in pre-transfusion hemoglobin levels in TD patients  
 To evaluate changes in markers of erythropoiesis, hemolysis, iron overload, and iron 
metabolism 


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 To examine the pharmacokinetic (PK) profile of ACE-536 in patients with β-thalassemia  
Exploratory: 
 To evaluate biomarkers related to the transforming growth factor beta (TGF-) superfamily 
 To evaluate patient self-reported quality of life using tools including but not limited to the 
Functional Assessment of Cancer Therapy-Anemia Scale (FACT-An) and Short Form (36) 
Health Survey (SF-36) questionnaires  
 To evaluate change in extramedullary hematopoiesis (EMH) mass size by MRI 
 To evaluate change in spleen size by MRI 
 To evaluate change in bone mineral density (BMD) by DXA 
 To evaluate change in leg ulcers  
 To evaluate change in the 6-minute walk test (6MWT) distance in NTD patients 
Methodology  
Study Design:  
This open-label extension study will evaluate the effects of up to 60 months of ACE-536 
treatment in patients with β-thalassemia previously enrolled and treated with ACE-536 for up to 
3 months in study A536-04.  The base study (A536-04) is a phase 2, open-label, ascending dose 
study to evaluate the effects of ACE-536 in patients with β-thalassemia.  A total of up to 
64 patients may be enrolled in study A536-04 and may be eligible for study A536-06.   
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.  For these patients, Cycle 1 
Day 1 (C1D1) of study A536-06 may take place 28 (± 7) days after the last dose administered 
in study A536-04, which may coincide with the patient’s A536-04 End of Treatment (EOT) 
visit.  These patients are considered “patients without treatment interruption”.     
Patients who complete the EOS visit for study A536-04 and are ≥ 28 days post EOS visit are 
considered “patients with treatment interruption” and will be re-assessed for eligibility by 
meeting all eligibility criteria plus additional inclusion criteria, as defined in Section 9.3.  
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 be treated as 
patients with treatment interruption and should not begin study A536-06 C1D1 until they have 
reached ≥ 28 days post EOS visit from study A536-04 so that new baseline assessments can be 
measured.  
For patients without treatment interruption, transfusion status (NTD or TD) will carry-over 
from the base study A536-04.  For NTD patients with treatment interruption, transfusion status 
will be reassessed prior to C1D1 of study A536-06.  NTD patients with treatment interruption 
are defined as patients who require transfusion of < 4 units of RBCs over the 8 weeks prior to 
Cycle 1 Day 1 of study A536-06.  For TD patients with treatment interruption, transfusion 
status will carry-over from the base study A536-04. 
A patient without treatment interruption may continue to be dosed with ACE-536 at the same 
dose level (rounded to nearest A536-06 starting dose level) administered at their last dose in 


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study A536-04 (unless a dose reduction is required based upon patient dose modification rules 
from study A536-06).  All patients with treatment interruption will be initially treated with 
ACE-536 at a starting dose level of 0.8 mg/kg which has been determined to be safe and well 
tolerated by the Safety Review Team (SRT) based on data from study A536-04.   
Each TD patient 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 of base 
study A536-04 (for patients without treatment interruption) or study A536-06 (for patients with 
treatment interruption).  During treatment, if the pre-transfusion hemoglobin level 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). 
Patients will participate in the extension study A536-06 for up to 97 months, including a 28-day 
(1 month) screening period, a 60-month treatment period and a 3-year (36 months) follow-up 
period. 
The SRT established for study A536-04 will review safety data and pharmacodynamic (PD) 
data periodically throughout study A536-06. 
Starting Dose Levels  
A patient without treatment interruption may continue to be dosed with ACE-536 at the same 
dose level (rounded to nearest A536-06 starting dose level) administered at their last dose in 
study A536-04 (unless a dose reduction is required based upon patient dose modification rules 
from study A536-06).  All patients with treatment interruption will be initially treated with 
ACE-536 at a starting dose level of 0.8 mg/kg which has been determined to be safe and well 
tolerated by the Safety Review Team (SRT) based on data from study A536-04.  Examples of 
possible starting dose levels with dose modifications (reductions and titrations) are below for 
reference.     
 




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a Patients who require more than 2 dose reductions due to an AE should be discontinued from treatment and 
complete the EOT, PTFU, LTFU, and EOS visits. 
Observation on Dosing Day 
Action 
ACE-536 Dose Modification 
Hemoglobin ≥ 13 g/dL 
Hold dose and monitor 
patient weekly until 
hemoglobin < 12 g/dL 
Resume dosing once hemoglobin 
< 12 g/dL at the same dose level 
Hemoglobin increase ≥ 2 g/dL 
from the previous dosing day 
(not attributable to RBC 
transfusion) 
Continue dosing 
Reduce the dose by 1 dose level 
Non-hematologic AE ≥ Grade 3, 
or hematologic AE ≥ 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 the dose by 1 dose levela 
Grade 3 leukocytosis (WBC 
>100,000/μL) 
 
Discontinue treatment 
Individual Dose Titration for NTD Patients 
For all NTD patients (with and without treatment interruption), the dose level will be assessed 
for titration at Day 1 of every cycle starting at C3D1 (e.g., C3D1, C4D1, C5D1, etc.).  The dose 
level should be titrated individually for each patient not to exceed the maximum dose level 
evaluated in the base study A536-04 as follows:   
 If hemoglobin increase from baseline is < 1.5 g/dL throughout the previous two 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 two 
consecutive measurements during the previous two 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 two consecutive 
measurements during the previous two 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 two or more measurements 
performed during the screening period for base study A536-04 (for patients without treatment 
interruption) or study A536-06 (for patients with treatment interruption).  Hemoglobin 
measurements within 2 weeks following RBC transfusion will be excluded from both the 
calculation of the mean baseline hemoglobin and for evaluation of dose titration for a patient 
during treatment.  
Individual Dose Titration for TD Patients 
For all TD patients (with and without treatment interruption), the dose level will be assessed for 
titration at Day 1 of every cycle starting at C3D1 (e.g., C3D1, C4D1, C5D1, etc.).  The dose 


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level should be titrated individually for each patient not to exceed the maximum dose level 
evaluated in the base study A536-04 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).   
Number of patients: A planned total of up to 64 patients who enrolled in base study A536-04 
may be eligible for the extension study A536-06.   
Inclusion Criteria: 
All patients must meet the following criteria 
1. Completion of the treatment period in the base study A536-04.   
2. 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. 
3. Patient is able to adhere to the study visit schedule, understand and comply with all 
protocol requirements. 
4. Patient understands and is able to provide written informed consent.  
Patients with treatment interruption (defined as patients who complete the EOS visit for 
study A536-04 and are ≥ 28 days post EOS visit) must also meet the following criteria 
5. Mean hemoglobin concentration < 10.0 g/dL of 2 measurements (not influenced by 
RBC transfusion) (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 NTD patients. 
6. Adequate folate levels or on folate therapy.   
7. Platelet count ≥ 100 x 109/L and ≤ 1,000 x 109/L.   
8. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) < 3 x upper limit 
of normal (ULN). 
9. Serum creatinine ≤ 1.5 x ULN.  
10. Ejection fraction ≥ 50% by Echocardiogram (ECHO) or Multi gated acquisition scan 
(MUGA).  
Exclusion Criteria: 
All patients must not meet any of the following criteria 
1. Discontinuation/withdrawal from study A536-04 due to patient request, patient 
unwillingness or inability to comply with the protocol, pregnancy, use of prohibited 


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medication (e.g., hydroxyurea), medical reason or AE, hypersensitivity reaction to the 
study drug, at the discretion of the sponsor, or loss to follow-up prior to completion of 
the treatment period.   
2. 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.  
3. Symptomatic splenomegaly.  
4. Splenectomy within 56 days prior to Cycle 1 Day 1. 
5. 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. 
6. Patients receiving or planning to receive hydroxyurea treatment.  Patients must not have 
had hydroxyurea within 90 days of Cycle 1 Day 1.    
7. For patients with treatment interruption: Iron chelation therapy if initiated within 
56 days prior to Cycle 1 Day 1. 
8. 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 and low molecular weight (LMW) heparin for superficial vein 
thrombosis (SVT) is permitted). 
9. Treatment with another investigational drug (including sotatercept [ACE-011]) 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 at any time between the end of 
treatment of the base study A536-04 and Cycle 1 Day 1.  
10. Known positive for human immunodeficiency virus (HIV), active infectious hepatitis B 
(HBV) or active infectious hepatitis C (HCV).  
11. Uncontrolled hypertension defined as systolic blood pressure (SBP) ≥ 150 mm Hg or 
diastolic blood pressure (DBP) ≥ 100 mm Hg.  
12. 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). 
13. Pregnant or lactating females. 
14. History of severe allergic or anaphylactic reactions or hypersensitivity to recombinant 
proteins or excipients in the investigational drug. 
15. Any other condition not specifically noted above which, in the judgment of the 
investigator, would preclude the patient from participating in the study.  


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Investigational product, dosage and mode of administration: 
ACE-536 drug product will be provided as either a sterile liquid formulation or a lyophilized 
powder formulation for reconstitution with water.  Each single-use vial of the liquid 
formulation contains 25 mg of ACE-536 in a 0.5 mL Tris-buffered saline solution (50 mg/mL).  
Each single-use vial of the lyophilic formulation may contain either 25 mg, 50 mg, or 75 mg 
ACE-536 for reconstitution with 0.5 mL, 1 mL, or 1.5 mL water, respectively, to form a citrate-
buffered solution (50 mg/mL) for injection. 
ACE-536 will be administered by SC injection on Day 1 of each cycle. 
Criteria for Evaluation 
Safety Assessments: 
 All patients will be assessed for safety by monitoring AEs, clinical laboratory tests, 
vital signs, electrocardiogram (ECG), anti-drug antibody (ADA) testing, abdominal 
MRI/ultrasound and physical examination.   
Efficacy Assessments:  
 Patients will be assessed for erythroid response over a 62-month period following 
initiation of treatment.  Erythroid response endpoints will be determined by 
monitoring hematologic laboratory values and RBC transfusions. 
 Other secondary efficacy endpoints will be assessed by examining other 
hematology, erythropoiesis, hemolysis, iron overload, and iron metabolism 
parameters. 
o Erythropoiesis parameters may include serum erythropoietin (EPO) levels, 
reticulocytes, nucleated RBCs and soluble transferrin receptor 
o Iron overload/metabolism parameters may include serum iron, total iron 
binding capacity (TIBC), transferrin, soluble transferrin receptor, calculated 
transferrin saturation, ferritin, non-transferrin bound iron (NTBI), hepcidin, 
Liver iron content (LIC), and iron chelation therapy use. 
PK Assessments: 
 Blood samples will be drawn to evaluate PK parameters. 
Exploratory Assessments: 
 Biomarkers related to the TGF-β superfamily 
 Self-reported quality of life measures including but not limited to the FACT-An and 
SF-36 questionnaires 
 Extramedullary hematopoiesis (EMH) mass size by MRI 
 
Spleen size by MRI 
 
Bone mineral density (BMD) by DXA 
 
Leg ulcer size 
 
6-minute walk test (6MWT) distance in NTD patients. 


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Statistical Methods  
Sample Size Calculation:  
There is no formal sample size calculation for the study although up to 64 patients may 
participate from the A536-04 study.   
Analysis Populations: 
The modified Intent-to-Treat (mITT) Population: All patients enrolled in the study who 
received at least one dose of ACE-536.  
Efficacy Evaluable (EE) Population: All patients administered at least one dose of ACE-536 
and have 1) at least three hemoglobin assessments over an 8-week period, not influenced by 
transfusion, in NTD patients, OR, 2) at least 8 consecutive weeks of transfusion frequency data 
in TD patients. 
Pharmacokinetics Population: All patients who received at least 1 dose of ACE-536 during 
Study A536-06 and have sufficient serum ACE-536 values for PK analysis. 
Primary Endpoint Analysis: 
Safety analysis: To assess clinical safety, all AEs, vital sign measurements and clinical 
laboratory information will be summarized.  AEs 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. Disposition rates will be summarized. 
Efficacy/Pharmacodynamic Effects Analysis of Secondary Endpoints:  
All efficacy analyses will be performed for the EE and mITT populations. 
Efficacy will be evaluated by determining the proportion of β-thalassemia patients who have an 
erythroid response, defined as 1) a mean hemoglobin increase ≥ 1.0 g/dL or ≥ 1.5 g/dL over an 
8-week or 12-week period as compared to baseline, not influenced by RBC transfusion in NTD 
patients or 2) ≥ 20% or ≥ 50% reduction in RBC transfusion burden over an 8-week or 12-week 
period as compared to the 8-week or 12-week period pretreatment in TD patients. 
Baseline hemoglobin for NTD patients will be the average of two or more measurements 
performed during the screening period for base study A536-04 (for patients without treatment 
interruption) or study A536-06 (for patients with treatment interruption).  Hemoglobin 
measurements within 2 weeks following RBC transfusion will be excluded. 
Transfusion burden for TD patients will be defined as the ratio of RBC transfusion units 
(or mLs) transfused during an interval divided by the duration 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 8 or 12 weeks prior to Cycle 1 
Day 1 of base study A536-04 (will also be analyzed using pretreatment period of study 
A536-06 for patients with treatment interruption).  An interval during the treatment plus 


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follow-up period will be defined as an 8-week or 12-week interval after Cycle 1 Day 1 of study 
A536-06. 
The erythroid response, as defined above, will also be evaluated using other pre-treatment and 
on-treatment intervals and other % reductions in RBC units transfused, including 100% 
(transfusion-free). 
The erythroid response will be summarized using both a point estimate and its exact 95% 
confidence interval (CI) based on binomial distribution. 
No direct comparison testing with concurrent or historical controls will be performed.   
Analysis of other secondary efficacy/PD endpoints will include: 
 Time to erythroid response and duration of erythroid response 
 Change/percent change from pretreatment in mean transfusion burden in TD 
patients 
 Change in mean hemoglobin level in NTD patients 
 Change in pre-transfusion hemoglobin levels in TD patients (including analysis of 
patient subset with < 20% change in transfusion burden)   
 Change from baseline in erythropoiesis parameters, including EPO, reticulocytes, 
nucleated RBCs, and soluble transferrin receptor 
 Change from baseline in hemolysis parameters, including total/indirect bilirubin, 
and lactate dehydrogenase (LDH)  
 Change from baseline in iron overload and iron metabolism parameters, including 
serum iron, TIBC, transferrin, calculated transferrin saturation, soluble transferrin 
receptor, ferritin, NTBI, hepcidin, iron chelation therapy use and LIC by MRI. 
The exploratory endpoints will include evaluation of biomarkers related to the TGF- 
superfamily, quality of life, change in EMH mass size, change in spleen size (in patient subset 
with no prior splenectomy), change in BMD, 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 CI 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% CI. 
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 ADA on ACE-536 PK and drug exposure if ADA tests 
are deemed positive. 
Pharmacokinetics analysis: PK parameters for ACE-536, such as maximum serum 
concentration (Cmax), time to maximum concentration (tmax), and area under the 
concentration/time curve (AUC), will be estimated using non-compartmental and/or single 
compartment analysis, as appropriate.  Dose proportionality may be assessed using the 
exposure data (e.g., Cmax, AUC) if sufficient dose levels are studied.  Descriptive statistics will 


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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, as appropriate. 








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1 
Screening procedures: Other than informed consent, procedures listed as part of the 28-day screening period are only applicable to patients with treatment interruption.  ECHO or 
MUGA (only required for patients with treatment interruption) can be performed up to 56 days prior to C1D1.  If performed as part of standard of care, ECHO/MUGA does not 
need to be repeated.  Patients with treatment interruption will need to qualify per the additional inclusion criteria listed in Section 9.3.2.  For patients without treatment interruption, 
C1D1 may coincide with EOT visit of the base study A536-04.  Procedures that are required to confirm eligibility for patients without treatment interruption can be performed at 
the base study A536-04 EOT visit and used to confirm eligibility prior to dosing on C1D1 of study A536-06.   
2 
Study procedures must be done prior to administration of study drug.  Note that all windows on visits should be determined relative to the date of the previous dose of ACE-536. 
For patients without treatment interruption, C1D1 procedures shaded grey may be conducted as part of the EOT visit for study A536-04 and may not need to be repeated for study 
A536-06.  All patients must be assessed for eligibility prior to dosing on C1D1.  C1D1 MRI for LIC (at selected sites) and EMH masses (unless site does not have feasibility to 
perform the assessment) and MRI of the spleen (if no prior splenectomy) can be performed up to 56 days prior to C1D1.  If performed as part of standard of care, MRI for LIC, 
MRI for EMH and spleen size do not need to be repeated.  All screening and C1D1 procedure results required to confirm eligibility must be obtained and reviewed prior to study 
drug administration in A536-06.  On dosing days: Note that the patient dose must be calculated based on the patient’s weight on the day of dosing.  Starting dose level for C1D1, 
dose modification rules and titration rules must be reviewed and implemented prior to dosing as required per protocol (see Section 10.7, Section 10.8 and Section 10.9).  If a dose 
delay is required per the dose modification rules (Section 10.8), the patient will not be dosed.  The patient will return weekly for assessment of hematology results and AEs until 
the patient is eligible to receive the next dose of ACE-536 and start the next cycle. The patient should resume the study at the planned dosing cycle (e.g. if the patient missed a dose 
at C4D1, then they would resume dosing at C4D1 and not skip to C5D1).    
3 
Medical history: Medical history for patients with treatment interruption will include medical events occurring after EOS visit in study A536-04 and prior to C1D1 for study 
A536-06.  Medical history for patients without treatment interruption will be taken from study A536-04. 
4 
Quality of life questionnaires are required at C1D1, C5D1, C9D1, C13D1 C17D1, C25D1, C33D1, C41D1, C49D1, C57D1, C65D1, C73D1, C81D1 and EOT. 
5 
Physical exam: Physical exam should also include an optional evaluation of gonadal size of male patients on C1D1, C5D1, C13D1, C22D1, C30D1, C38D1, C46D1, C54D1, 
C62D1, C70D1, C78D1 EOT and EOS. 
6 
Vital signs will include weight, heart rate, and SBP and DBP. 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. 
7 
MRI for liver iron content will be performed at selected sites at C1D1, C9D1 C17D1, C25D1, C33D1, C41D1, C49D1, C57D1, C65D1, C73D1, C81D1 and EOT within 
a +/- 10-day window for each scan. A MRI for liver iron content is not required if < 3 months since the previous MRI for liver iron content, unless clinically indicated.  
8 
MRI of the chest and abdomen for EMH mass measurement will be performed for at selected sites at C1D1. If the assessment at C1D1 is negative (no masses), 
additional scans are not required unless clinically indicated. If the assessment at C1D1 is positive (masses present), repeat MRI at C9D1, C17D1, C25D1, C33D1, C49D1, 
C65D1, C81D1 and EOT within a +/- 10-day window for each scan. A MRI of the chest and abdomen for EMH mass measurement is not required if < 3 months since the 
previous MRI of the chest and abdomen for EMH mass measurement, unless clinically indicated 
9 
MRI of the spleen: will be performed at selected sites at C1D1 on patients with no prior splenectomy. If there is no indication of an enlarged spleen on the C1D1 scan, 
additional scans are not required unless clinically indicated. If there is an enlarged spleen on the C1D1 scan, repeat imaging at C9D1 C17D1, C25D1 C33D1, C49D1, 
C65D1, C81D1 and EOT within a +/- 10-day window for each scan.  Patients with clinical signs of a change in spleen size or abnormality should have an abdominal 
ultrasound or MRI performed as needed throughout the study. A MRI for the spleen is not required if < 3 months since the previous MRI for the spleen, unless clinically 
indicated.     
10 
Leg ulcer assessment: Patients with leg ulcers should have regular assessment of the leg ulcer(s) throughout the study.  Photographs of the leg ulcer(s) pre- and post- dose 
should be provided when available to document any changes in the ulcer(s).   
11 
Serum iron studies: May include serum iron, TIBC, transferrin, soluble transferrin receptor, calculated transferrin saturation, ferritin, NTBI.  The sponsor may perform 
additional sample analysis for biomarkers for exploratory research purposes only. 
12 
Serum folate: Required at screening for patients with treatment interruption to confirm eligibility.  Required for all patients at C1D1, C5D1, C13D1, C21D1, C29D1 
C37D1, C45D1, C53D1, C61D1, C69D1, C77D1, and EOT. 
13 
Hematology: RBC, nucleated red blood cells (nRBC) (local and central lab), white blood cell (WBC) with differential, hemoglobin, hematocrit, reticulocyte count, platelet 
count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution 
width (RDW).  On dosing days, hemoglobin values are to be drawn and resulted (up to 24 hours) prior to dosing (see Section 10.8, Patient Dose Modification Rules).  
14 
Peripheral blood smear: Peripheral blood smears will be prepared centrally from blood sample. 


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15 
Chemistry: Sodium, potassium, AST, ALT, 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.  Perform at screening, C1D1, C3D1, C5D1, C9D1, C13D1, C17D1, C21D1, C25D1, C29D1, 
C33D1, C37D1, C41D1, C45D1, C49D1, C53D1, C57D1, C61D1, C65D1, C69D1, C73D1, C77D1, C81D1, EOT and EOS. 
16 
Urinalysis by dipstick analysis (local lab): pH, specific gravity, protein, glucose, ketones, blood, leukocyte esterase, and nitrite, with microscopic examination if 
indicated.  Urine chemistry (central lab): urine chemistries include but are not limited to: microalbumin and creatinine. 
17 
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. 
18 
PK collection is only required on C1D1, C1D8, C2D1, C9D1, C9D8, C10D1, C17D1, and C17D8. 
19 
PD biomarkers: May include hepcidin, GDF15, GDF8, GDF11, activin A and others to be determined.  The sponsor may perform additional sample analysis for biomarkers for 
exploratory research purposes only. 
20 
Hemoglobin electrophoresis: Samples will be collected for central hemoglobin electrophoresis on C1D1, C5D1, C13D1, C21D1, C29D1, C37D1, C45D1, C53D1, 
C61D1, C69D1, C77D1, EOT, and EOS. 
21 
Pregnancy test: (urine or serum) is required for female patients of child bearing potential only. 
22 
Menstrual history: is required for female patients of child bearing potential only 
23 
Transfusion frequency/volume: Transfusion history will be collected from the EOS visit in study A536-04 through the C1D1 visit of A536-06 as available; TD patients 
will have a “pre-transfusion hemoglobin threshold” for requiring transfusion during the study which will be determined based on transfusion history.  For TD patients 
without treatment interruption, baseline pre-transfusion hemoglobin threshold will be the mean of all documented pre-transfusion hemoglobin values during the 12 weeks 
prior to C1D1 of base study A536-04 or study A536-06 (for TD patients with treatment interruption).  During treatment, if the pre-transfusion hemoglobin level 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). 
24 
Adverse events: Patients should be monitored for AEs throughout the study. 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. 
25 
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 EOT 
visit at the time of discontinuation.   
26 
Post-Treatment Follow-Up (PTFU): Should be performed 2 months (± 7 days) after the last dose of ACE-536.  
27 
Long-Term Follow-Up (LTFU): Should be performed every 6 months (± 14 days) after the last dose of ACE-536 for 3 years after the last dose of ACE-536 to monitor for 
presence of malignancy and pre-malignancy, as per standard of care.  
28 
End of Study (EOS): Should be performed 3 years after the last dose of ACE-536.     
29 
Cycles 6 through 17: Procedures and visits for Cycles 6 through 9, 10 through 13, and 14 through 17 follow the same procedures and visits as scheduled for Cycles 2 
through 5, with some exceptions as noted above. 
30 
Cycles 24 through 87: Patients may not exceed 87 cycles or 1825 days after first dose, whichever occurs first. The last dose of ACE-536 may not be administered after 
Day 1825, regardless of the number of cycles completed. Procedures and visits for Cycles 24 through 27, 28 through 31, 32 through 35, 36 through 39, 40 through 43, 44 
through 47, 48 through 51, 52 through 55, 56 through 59, 60 through 63, 64 through 67, 68 through 71, 72 through 75, 76 through 79, 80 through 83 and 84 through 87 
follow the same procedures and visits as scheduled for Cycles 20 through 23, with some exceptions as noted above. 
31 
DXA for BMD will be performed at selected sites at C1D1, C9D1, C17D1, C25D1, C33D1, C49D1, C65D1, C81D1 and EOT within a +/- 10-day window for each scan. 
A DXA for BMD is not required if < 3 months since the previous DXA for BMD, unless clinically indicated.     
32 
6MWT for NTD patients will be performed at selected sites at C1D1, C5D1, C9D1, C13D1, C17D1, C25D1, C33D1, C41D1, C49D1, C57D1, C65D1, C73D1, C81D1 
and EOT.  
 


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3. 
TABLE OF CONTENTS 
1. 
PROTOCOL SYNOPSIS .............................................................................................6 
2. 
SCHEDULE OF EVENTS .........................................................................................17 
3. 
TABLE OF CONTENTS ...........................................................................................22 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................26 
5. 
ETHICS ......................................................................................................................29 
5.1. 
Institutional Review Board .........................................................................................29 
5.2. 
Ethical Conduct of the Study ......................................................................................29 
5.3. 
Patient Information and Consent ................................................................................29 
5.4. 
Patient Data Protection ...............................................................................................29 
6. 
INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE .................30 
7. 
INTRODUCTION ......................................................................................................31 
7.1. 
Overview of Target Indication ....................................................................................31 
7.2. 
Summary of Nonclinical Studies ................................................................................32 
7.2.1. 
Pharmacology Studies ................................................................................................32 
7.2.2. 
Toxicology Studies .....................................................................................................33 
7.3. 
Summary of Clinical Experience ................................................................................33 
7.4. 
Potential Risks of Human Use ....................................................................................34 
8. 
TRIAL OBJECTIVES ................................................................................................35 
8.1. 
Primary Objectives .....................................................................................................35 
8.2. 
Secondary Objectives .................................................................................................35 
8.3. 
Exploratory Objectives ...............................................................................................35 
9. 
OVERALL STUDY DESIGN AND PLAN: DESCRIPTION ..................................36 
9.1. 
Study Design ...............................................................................................................36 
9.2. 
Discussion of Study Design ........................................................................................37 
9.3. 
Selection of Study Population ....................................................................................37 
9.3.1. 
Transfusion Status ......................................................................................................37 
9.3.2. 
Inclusion Criteria ........................................................................................................38 
9.3.3. 
Exclusion Criteria .......................................................................................................38 
9.4. 
Patient Treatment Discontinuation and Withdrawal Criteria .....................................39 
10. 
TREATMENT OF PATIENTS ..................................................................................41 
10.1. 
Selection and Timing of Dose for Each Patient ..........................................................41 


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10.2. 
Concomitant Medications ...........................................................................................41 
10.2.1. 
Prohibited Concomitant Medications and Procedures ................................................41 
10.2.2. 
Other Treatments for β-Thalassemia ..........................................................................41 
10.2.3. 
RBC Transfusions .......................................................................................................42 
10.3. 
Treatment Compliance ................................................................................................42 
10.4. 
Randomization ............................................................................................................42 
10.5. 
Treatments Administered ............................................................................................42 
10.6. 
Safety Evaluation ........................................................................................................42 
10.7. 
Starting Dose Levels ...................................................................................................43 
10.8. 
Individual Dose Modification Rules ...........................................................................43 
10.9. 
Individual Dose Titration ............................................................................................44 
10.9.1. 
Individual Dose Titration for NTD Patients ...............................................................44 
10.9.2. 
Individual Dose Titration for TD Patients ..................................................................44 
10.10. 
Other Considerations for Dose Modification, Delay or Discontinuation ...................45 
11. 
STUDY PROCEDURES ............................................................................................46 
11.1. 
Written Informed Consent ..........................................................................................46 
11.2. 
Clinical Laboratory Tests ...........................................................................................46 
11.2.1. 
Local Laboratory Testing ...........................................................................................46 
11.2.2. 
Central Laboratory Testing .........................................................................................46 
11.3. 
Other Safety Assessments ...........................................................................................47 
11.4. 
Pharmacokinetic and Pharmacodynamic Assessments ..............................................47 
11.4.1. 
Pharmacokinetic Assessments ....................................................................................47 
11.4.2. 
Pharmacodynamic Evaluations ...................................................................................47 
12. 
STUDY SCHEDULE .................................................................................................49 
12.1. 
Screening ....................................................................................................................49 
12.2. 
Dosing Days and Interim Visits ..................................................................................49 
12.3. 
End of Treatment Visit ...............................................................................................51 
12.4. 
Post-Treatment Follow-Up Visit ................................................................................51 
12.5. 
Long-Term Follow-Up Visits .....................................................................................51 
12.6. 
End of Study Visit ......................................................................................................51 
12.7. 
Termination of Study ..................................................................................................51 
13. 
STUDY DRUG MATERIALS AND MANAGEMENT ...........................................52 
13.1. 
Study Drug ..................................................................................................................52 


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13.2. 
Study Drug Packaging and Labeling ..........................................................................52 
13.3. 
Study Drug Storage .....................................................................................................52 
13.4. 
Study Drug Preparation ..............................................................................................52 
13.5. 
Study Drug Administration.........................................................................................52 
13.6. 
Study Drug Accountability .........................................................................................52 
13.7. 
Study Drug Handling and Disposal ............................................................................53 
14. 
ASSESSMENT OF SAFETY .....................................................................................54 
14.1. 
Adverse Event Definitions ..........................................................................................54 
14.2. 
Pregnancy and In Utero Drug Exposure .....................................................................55 
14.3. 
Severity .......................................................................................................................56 
14.4. 
Relationship to Study Drug ........................................................................................56 
14.5. 
Documentation and Methods of Reporting of Adverse Events by Investigator .........56 
14.5.1. 
Documentation of Serious Adverse Events ................................................................57 
14.6. 
Reporting Period and Monitoring of Patients with Adverse Events ...........................57 
14.7. 
Notification about Serious Adverse Events ................................................................58 
14.7.1. 
Safety Reporting to Health Authorities, Independent Ethics Committees 
Institutional Review Boards and Investigators ...........................................................58 
15. 
STATISTICS ..............................................................................................................60 
15.1. 
Analysis Populations ..................................................................................................60 
15.2. 
Statistical Plan ............................................................................................................60 
15.2.1. 
Patient Accountability and Demographics .................................................................60 
15.2.2. 
Primary Endpoint Analysis .........................................................................................60 
15.2.3. 
Secondary Analysis ....................................................................................................60 
15.2.4. 
Pharmacokinetics Analysis .........................................................................................62 
15.2.5. 
Anti-drug Antibody Analysis .....................................................................................62 
15.3. 
Determination of Sample Size ....................................................................................62 
15.4. 
Interim Analysis ..........................................................................................................62 
15.5. 
Deviation from Original Analysis Plan ......................................................................62 
16. 
SOURCE DOCUMENTATION AND INVESTIGATOR FILES .............................63 
16.1. 
Study Monitoring ........................................................................................................63 
16.2. 
Audits and Inspections ................................................................................................63 
17. 
QUALITY CONTROL AND QUALITY ASSURANCE .........................................64 
17.1. 
Data Quality Control and Quality Assurance .............................................................64 


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17.1.1. 
Investigator Responsibility .........................................................................................64 
17.1.2. 
Protocol Modifications ...............................................................................................64 
18. 
CONFIDENTIALITY ................................................................................................65 
19. 
PROTOCOL AMENDMENTS ..................................................................................66 
20. 
DATA HANDLING AND RECORDKEEPING .......................................................67 
20.1. 
Case Report Form Completion ...................................................................................67 
20.2. 
Retention of Records ..................................................................................................67 
21. 
STUDY FINANCE AND INSURANCE ...................................................................68 
21.1. 
Study Finance .............................................................................................................68 
21.2. 
Insurance .....................................................................................................................68 
22. 
REFERENCES ...........................................................................................................69 
23. 
APPENDICES ............................................................................................................70 
23.1. 
Appendix 1: National Cancer Institute (NCI) Common Terminology Criteria 
for Adverse Events (CTCAE) .....................................................................................70 


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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 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CXDY 
Cycle X Day Y 
CI 
Confidence interval 
Cmax 
Maximum concentration 
CREAT 
Creatinine 
CRF 
Case report form 
CRO 
Contract research organization 
DBP 
Disastolic blood pressure 
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 
EPO 
Erythropoietin 
EOT 
End of treatment 
FACT-An 
Functional Assessment of Cancer Therapy-Anemia Scale 
FDA 
United States Food and Drug Administration 


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Abbreviation 
Definition 
FSH 
Follicle stimulating hormone 
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 
LIC 
Liver iron content 
LDH 
Lactate dehydrogenase 
LMW 
Low molecular weight 
LTFU 
Long-term follow-up 
MedDRA 
Medical Dictionary for Regulatory Activities 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
miTT 
Modified intent-to-treat 
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  
NTD 
Non-transfusion dependent 
PD 
Pharmacodynamic 


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Abbreviation 
Definition 
PHI 
Protected health information 
PK 
Pharmacokinetic 
PTFU 
Post-treatment follow-up 
QoL 
Quality of life 
RBC 
Red blood cell 
RDW 
Red blood cell distribution width 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SBP 
Systolic blood pressure 
SC 
Subcutaneous 
SD 
Standard deviation 
SRT 
Safety review team 
SF-36 
Short Form (36) Health Survey 
SUSAR 
Suspected unexpected serious adverse reaction 
SVT 
Superficial venous thrombosis 
TD 
Transfusion dependent 
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 Independent Ethics Committee 
(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 patient.  
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, 
(erythropoiesis).  In nonclinical experiments, ACE-536 has been shown to bind with high affinity 
to some TGF-β ligands (e.g., GDF8, GDF11, BMP6 and activin B) but substantially less, or not 
at all, to others (e.g., BMP10 and activin A).  The emerging body of evidence on ACE-536 
suggests that its mechanism of action is completely independent from that of erythropoietin 
(EPO), and involves stimulation of the later, maturation phase of erythroblast differentiation and 
maturation in the bone marrow. 
7.1. 
Overview of Target Indication 
The target indication for ACE-536 is for the treatment of 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  
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 
02 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.6  Children with β-thalassemia who are periodically 
transfused to maintain a hemoglobin value of 9.5 to 14 g/dL grow and develop normally.  
However, these patients require lifelong transfusion support, and concomitant iron chelation 


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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 nonclinical pharmacology studies have been conducted with ACE-536 or its 
murine ortholog, RAP-536.  The RAP-536 molecule has the same ECD as ACE-536, but 
contains a murine IgG Fc domain in place of the human IgG Fc.  The RAP-536 ortholog is 
substantially less immunogenic in rodent species and therefore permitted the conduct of longer 
term pharmacology studies in rodents without the confounding influence of immune reactivity. 
ACE-536 has been shown to have significant effects on erythropoiesis.  Evidence suggests that it 
binds ligands that normally act to modulate the differentiation of late-stage erythroid precursors 
(normoblasts).  Inhibition of these ligands leads to a rapid and robust erythroid response and 
increased production of mature erythrocytes.  Because ACE-536 affects late-stage erythrocyte 
precursor populations, its action is distinct from that of EPO.  Unlike EPO, ACE-536 is not a 
growth-promoting cytokine. 
Studies of ACE-536 in normal animals across several species (mice, rats, and cynomolgus 
monkeys) demonstrated an erythroid response that was rapid in onset, robust, and sustained.  
These effects were also demonstrated in a variety of animal models of anemia, including anemia 
associated with kidney failure, acute blood loss, and chemotherapy.   
RAP-536 was shown to significantly improve hematologic parameters, including increased 
hemoglobin concentrations, and to correct ineffective erythropoiesis and anemia in mouse 
models of myelodysplastic syndromes (MDS) and -thalassemia, both target indications for 
ACE-536.  This is consistent with its proposed mechanism of action of driving the late stages of 
erythroid differentiation that is downstream and distinct from EPO.  
The Hbb major-/- transgenic mouse was used to test RAP-536 in a model of -thalassemia.  This 
model represents human thalassemia intermedia, which is characterized by severe anemia, 
failure of erythroid differentiation, increased apoptosis of erythroid precursors, erythroid 
hyperplasia, splenomegaly and iron overload.  Treatment with RAP-536 led to significant 
improvement in hematological parameters, including RBC and hemoglobin.  Further effects of 


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treatment included decreased reticulocytes, red cell distribution width, EPO, bilirubin, RBC 
inclusion bodies, reactive oxygen species, serum ferritin, liver iron content (LIC), and spleen 
size, as well as increased hepcidin and bone mineral density, and improvements in RBC 
morphology. 
Additional information regarding pharmacological effects of ACE-536 is summarized in the 
current version of the ACE-536 IB. 
7.2.2. 
Toxicology Studies 
Repeat-dose toxicology studies of 1 and 3 month treatment duration have been conducted with 
ACE-536 in Sprague-Dawley rats and cynomolgus monkeys to evaluate the toxicity of 
ACE-536.  Recovery periods of up to 10 weeks were included as part of these studies.  In 
addition, the main phase of a repeat-dose toxicology study of 6 months treatment duration in 
cynomolgus monkeys is complete.  Findings from toxicology studies are described in more detail 
in the IB. 
7.3. 
Summary of Clinical Experience 
The safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) 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 PK parameters and PD effects of ACE-536.   
A total of 32 subjects were enrolled.  Eligible subjects were healthy, postmenopausal women, 
45-75 years old.  The anticipated PD effect was an increase in plasma hemoglobin level.  
Screening and baseline hemoglobin values had to be between 11.0 and 14.5 g/dL, inclusive, to 
mitigate the risk of substantial excursions above the normal range.  Sequential cohorts of 8 
subjects each were randomized to receive either ACE-536 (n=6) or placebo (n=2) administered 
as a SC injection on Day 1 and Day 15.  Each subject was to be followed for 112 days 
(16 weeks) after receiving their last dose of ACE-536 or placebo.  The following dose levels 
were evaluated: 0.0625, 0.125, and 0.25 mg/kg.  
ACE-536 administered every 2 weeks by subcutaneous (SC) injection for one or two doses at 
dose levels of 0.0625, 0.125 or 0.25 mg/kg was generally safe and well-tolerated in healthy 
postmenopausal female subjects.  No serious or severe adverse events (AE) were reported.  The 
majority of AEs were considered mild in severity.  AEs that were considered at least possibly 
drug-related across ACE-536 dose groups included injection site hemorrhage (3 subjects), 
injection site macule (2 subjects), and dry skin, macule, hyperesthesia, muscle spasms, myalgia, 
generalized pruritus, and papular rash (1 subject each).  Injection site pain was reported in one 
placebo-treated subject.  
PK results indicated mean AUC and Cmax values increased in a dose-proportional manner with 
ACE-536 dose level; mean Tmax ranged from 7.0 to 9.8 days, and mean T½ ranged from 14.9 to 
16.2 days after the first dose.  These PK results support intermittent SC dosing of ACE-536 in 
future studies, e.g., every 3 weeks. 
PD assessments for hematological markers showed increases in hemoglobin levels at the 
0.125 mg/kg dose level and more consistently at the 0.25 mg/kg dose level.  Small increases in 


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reticulocyte count and EPO levels were also observed at the two higher dose levels of ACE-536 
investigated.  A small decrease in serum follicle stimulating hormone (FSH) levels was observed 
at 0.25 mg/kg.  These data support further studies of ACE-536 in conditions associated with 
ineffective erythropoiesis and anemia. 
Two phase 2 studies with ACE-536 are ongoing.  Study A536-03 is entitled, “A Phase 2, 
Open-Label, Ascending Dose Study of ACE-536 for the Treatment of Anemia in Patients with 
Low or Intermediate-1 Risk Myelodysplastic Syndromes (MDS)”.  Study A536-04 is entitled “A 
Phase 2, Open-Label, Ascending Dose Study to Evaluate the Effects of ACE-536 in Patients with 
ß-Thalassemia”.  Preliminary results from these studies and additional information regarding the 
completed Phase 1 study can be found in the current version of the IB. 
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 patients and dose escalation/reduction rules will be utilized to minimize risks 
associated with increased RBC parameters. 
AEs observed in the Phase 1 study in healthy volunteers and the ongoing phase 2 studies that 
were considered probably or possibly related to study drug included injection site reactions 
(hemorrhage, pruritus, rash), skin rash, hyperesthesia, muscle spasms, myalgia, pruritus, and 
hyperkalemia. 
As with all biologics, there is the potential for anti-drug antibodies (ADA) that can be associated 
with increased drug clearance and hypersensitivity reactions.  ADA formation against ACE-536 
as well as human ActRIIB protein will be monitored in the initial clinical studies. 
Repeat-dose toxicology studies of 1 and 3 month treatment duration have been conducted with 
ACE-536 in Sprague-Dawley rats and cynomolgus monkeys to evaluate the toxicity of 
ACE-536.  In addition, the main phase of a repeat-dose toxicology study of 6 months treatment 
duration in cynomolgus monkeys is complete.  Reproductive toxicity studies are currently 
ongoing with ACE-536 and therefore ACE-536 should not be administered to pregnant or 
nursing 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 treatment and for at least 12 weeks following the last dose of ACE-536.  Please refer to 
the Investigator’s Brochure for additional information regarding findings from toxicology 
studies.  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 AE 
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 IB.  The ACE-536 IB should be reviewed prior to initiating the study. 


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8. 
TRIAL OBJECTIVES 
8.1. 
Primary Objectives 
 To evaluate the long-term safety and tolerability of ACE-536 in patients with 
β-thalassemia who were previously enrolled in study A536-04. 
8.2. 
Secondary Objectives 
 To evaluate erythroid response defined as the proportion of patients with: 
o Mean hemoglobin increase ≥ 1.5 g/dL over a continuous 12-week interval 
compared to baseline in non-transfusion dependent (NTD) patients, not 
influenced by RBC transfusion, OR 
o Reduction in RBC transfusion burden by ≥ 50% over a continuous 12-week 
interval compared to the 12 weeks prior to the start of treatment in transfusion 
dependent (TD) patients. 
 To evaluate erythroid response defined as proportion of patients with: 
o Mean hemoglobin increase ≥ 1.5 g/dL over a continuous 8-week interval 
compared to baseline in NTD patients, not influenced by RBC transfusion, 
OR 
o Reduction in RBC transfusion burden by ≥ 20% over a continuous 8-week 
interval compared to the 8 weeks prior to the start of treatment in TD patients. 
 To evaluate the time to erythroid response and duration of erythroid response. 
 To evaluate the mean change from baseline over an 8- or 12-week period in 
hemoglobin level in NTD patients not influenced by RBC transfusion 
 To evaluate the mean % change from baseline in transfusion burden over an 8- or 
12-week period in TD patients. 
 To evaluate the mean change in pre-transfusion hemoglobin levels in TD patients  
 To evaluate changes in markers of erythropoiesis, hemolysis, iron overload, and iron 
metabolism 
 To examine the PK profile of ACE-536 in patients with β-thalassemia 
8.3. 
Exploratory Objectives 
 To evaluate biomarkers related to the TGF- superfamily 
 To evaluate patient self-reported quality of life using tools including but not limited 
to the Functional Assessment of Cancer Therapy-Anemia Scale (FACT-An) and 
Short Form (36) Health Survey (SF-36) questionnaires  
 To evaluate change in extramedullary hematopoiesis (EMH) mass size by MRI 
 To evaluate change in spleen size by MRI 
 To evaluate change in bone mineral density (BMD) by DXA 


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 To evaluate change in leg ulcers  
 To evaluate change in the 6-minute walk test (6MWT) distance in NTD patients 
9. 
OVERALL STUDY DESIGN AND PLAN: DESCRIPTION  
This open-label extension study will evaluate the effects of up to 60 months of ACE-536 
treatment in patients with β-thalassemia previously enrolled and treated with ACE-536 for up to 
3 months in study A536-04.   
9.1. 
Study Design  
The base study (A536-04) is a phase 2, open-label, ascending dose study to evaluate the effects 
of ACE-536 in patients with β-thalassemia.  A total of up to 64 patients may be enrolled in study 
A536-04 and may be eligible for study A536-06.   
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.  For these patients, Cycle 1 Day 
1 (C1D1) of study A536-06 may take place 28 (± 7) days after the last dose administered in 
study A536-04, which may coincide with the patient’s A536-04 End of Treatment (EOT) visit.  
These patients are considered “patients without treatment interruption”.     
Patients who complete the EOS visit for study A536-04 and are ≥ 28 days post EOS visit are 
considered “patients with treatment interruption” and will be re-assessed for eligibility by 
meeting all eligibility criteria plus additional inclusion criteria, as defined in Section 9.3.  
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 be treated as 
patients with treatment interruption and should not begin study A536-06 C1D1 until they have 
reached ≥ 28 days post EOS visit from study A536-04 so that new baseline assessments can be 
measured.  
For patients without treatment interruption, transfusion status (NTD or TD) will carry-over from 
the base study A536-04.  For NTD patients with treatment interruption, transfusion status will be 
reassessed prior to C1D1 of study A536-06.  NTD patients with treatment interruption are 
defined as patients who require transfusion of < 4 units of RBCs over the 8 weeks prior to Cycle 
1 Day 1 of study A536-06.  For TD patients with treatment interruption, transfusion status will 
carry-over from the base study A536-04. 
A patient without treatment interruption may continue to be dosed with ACE-536 at the same 
dose level (rounded to nearest A536-06 starting dose level) administered at their last dose in 
study A536-04 (unless a dose reduction is required based upon patient dose modification rules 
from study A536-06).  All patients with treatment interruption will be initially treated with 
ACE-536 at a starting dose level of 0.8 mg/kg which has been determined to be safe and well 
tolerated by the Safety Review Team (SRT) based on data from study A536-04.   
Each TD patient 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 of base study 


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A536-04 (for patients without treatment interruption) or study A536-06 (for patients with 
treatment interruption).  During treatment, if the pre-transfusion hemoglobin level 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). 
Patients will participate in the extension study A536-06 for up to 97 months, including a 28-day 
(1 month) screening period, a 60-month treatment period and a 3-year (36 months) follow-up 
period. 
The SRT established for study A536-04 will review safety data and PD data periodically 
throughout study A536-06. 
9.2. 
Discussion of Study Design 
The primary endpoint is evaluating the long-term safety and tolerability of ACE-536 
administration in patients with β-thalassemia who completed participation in A536-04.  To 
support this endpoint, this study includes safety measurements that are considered standard for 
studies with investigational medications.   
An open-label study, conducted in a limited number of patients, with protocol-specified 
procedures for dose reduction is considered appropriate for a long-term safety study in this target 
patient population.  Individualization of dose level, using dose titration rules based on 
hemoglobin response and dose reduction rules based on safety information will maximize the 
proportion of patients having an erythroid response while maintaining safety. 
Secondary endpoints were selected to evaluate the impact of ACE-536 on efficacy and PD 
parameters, including effects on hematologic parameters and on complications of thalassemia. 
The initial dose level of 0.8 mg/kg has been selected for patients with treatment interruption 
based upon data from the ongoing A536-04 study.  Dose levels utilized in this study will not 
exceed maximum dose levels tested in study A536-04.   
9.3. 
Selection of Study Population 
9.3.1. 
Transfusion Status 
 For patients without treatment interruption patient transfusion status (NTD or TD) 
will carry-over from the base study A536-04.   
 For NTD patients with treatment interruption, patient transfusion status will be 
reassessed prior to C1D1 of study A536-06.   
 NTD patients with treatment interruption are defined as patients who require 
transfusion of < 4 units of RBCs over the 8 weeks prior to Cycle 1 Day 1 of study 
A536-06.   
 For TD patients with treatment interruption, patient transfusion status will carry-over 
from the base study A536-04.  


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9.3.2. 
Inclusion Criteria 
All patients must meet the following criteria: 
1. Completion of the treatment period in the base study A536-04.   
2. 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. 
3. Patient is able to adhere to the study visit schedule, understand and comply with all 
protocol requirements. 
4. Patient understands and is able to provide written informed consent.  
All patients with treatment interruption must also meet the following criteria: 
Patients with treatment interruption (defined as patients who complete the EOS visit for study 
A536-04 and are ≥ 28 days post EOS visit) must also meet the following criteria: 
5. Mean hemoglobin concentration < 10.0 g/dL of 2 measurements (not influenced by RBC 
transfusion) (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 NTD patients. 
6. Adequate folate levels or on folate therapy.   
7. Platelet count ≥ 100 x 109/L and ≤ 1,000 x 109/L.   
8. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) < 3 x upper limit of 
normal (ULN). 
9. Serum creatinine ≤ 1.5 x ULN.  
10. Ejection fraction ≥ 50% by Echocardiogram (ECHO) or Multi gated acquisition scan 
(MUGA).   
9.3.3. 
Exclusion Criteria 
All patients must not meet any of the following criteria: 
1. Discontinuation/withdrawal from study A536-04 due to patient request, patient 
unwillingness or inability to comply with the protocol, pregnancy, use of prohibited 
medication (e.g., hydroxyurea), medical reason or AE, hypersensitivity reaction to the 
study drug, at the discretion of the sponsor, or loss to follow-up prior to completion of the 
treatment period.   
2. Any clinically significant pulmonary (including pulmonary hypertension), 
cardiovascular, endocrine, neurologic, hepatic, gastrointestinal, infectious, 
immunological (including clinically significant allo- or auto-immunization) or 


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genitourinary disease considered by the investigator as not adequately controlled prior to 
Cycle 1 Day 1.  
3. Symptomatic splenomegaly.  
4. Splenectomy within 56 days prior to Cycle 1 Day 1. 
5. 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. 
6. Patients receiving or planning to receive hydroxyurea treatment.  Patients must not have 
had hydroxyurea within 90 days of Cycle 1 Day 1.    
7. For patients with treatment interruption: Iron chelation therapy if initiated within 56 days 
prior to Cycle 1 Day 1. 
8. 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 and low molecular weight (LMW) heparin for superficial venous 
thrombosis (SVT) is permitted). 
9. Treatment with another investigational drug (including sotatercept [ACE-011]) 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 at any time between the end of treatment of 
the base study A536-04 and Cycle 1 Day 1.  
10. Known positive for human immunodeficiency virus (HIV), active infectious hepatitis B 
(HBV) or active infectious hepatitis C (HCV).  
11. Uncontrolled hypertension defined as systolic blood pressure (SBP) ≥ 150 mm Hg or 
diastolic blood pressure (DBP) ≥ 100 mm Hg.  
12. 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). 
13. Pregnant or lactating females. 
14. History of severe allergic or anaphylactic reactions or hypersensitivity to recombinant 
proteins or excipients in the investigational drug.  
15. Any other condition not specifically noted above which, in the judgment of the 
investigator, would preclude the patient from participating in the study. 
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 


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 Pregnancy 
 Use of prohibited medication (e.g. hydroxyurea) 
 Medical reason or AE, 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 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 an SC 
injection on Cycle 1 Day 1.  Subsequent doses will be administered every 3 weeks on Day 1 of 
the cycle for up to 87 cycles.  Patients may not exceed 87 cycles or 1825 days after first dose, 
whichever occurs first.  The last dose of ACE-536 may not be administered after Day 1825, 
regardless of the number of cycles completed.  Dose reductions may be required for individual 
patients as outlined in the Patient Dose Modifications Rules (Section 10.8).  If a dose delay is 
required, the patient will return weekly for assessment of hematology results and AEs 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.  Dose level titration increases may also occur for individual patients as outlined in 
the Individual Dose Titration rules (Section 10.9).  If a patient has a positive 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).  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.  For patients without 
treatment interruption, any concomitant medication ongoing at the time of EOT in A536-04 
needs to be re-entered as a concomitant medication in A536-06.  For patients with treatment 
interruption, 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. 
Prohibited Concomitant Medications and Procedures 
No concurrent treatments with any other investigational agent are allowed.  
The following are not allowed during the treatment period: hematopoietic growth factors, 
anticoagulant therapies for deep vein thrombosis (DVT), and platelet aggregation inhibitors (with 
the exception of aspirin and LMW heparin). If a patient requires use of these therapies, the 
patient should be discontinued from the study treatment.  
10.2.2. 
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 EOT visit and enter the 
follow-up period.  Iron chelation therapy is allowed if initiated at least 56 days prior to C1D1.  If 
iron chelation therapy is required during study treatment, patient should follow standard of care 
for management of iron chelation therapy. 


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10.2.3. 
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 comorbidity. 
For any RBC transfusions (NTD or TD patients) received during the study, collect hemoglobin 
values just prior to transfusion. 
Each TD patient 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 of base study 
A536-04 (for patients without treatment interruption) or study A536-06 (for patients with 
treatment interruption).  During treatment, if the pre-transfusion hemoglobin level 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).  Patients may 
be transfused on the same day as ACE-536 is administered except on C1D1 for patients with 
treatment interruptions.  
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 study does not require randomization and is open-label. 
10.5. 
Treatments Administered 
A patient without treatment interruption may continue to be dosed with ACE-536 at the same 
dose level (rounded to nearest A536-06 starting dose level) administered at their last dose in 
study A536-04 (unless a dose reduction is required based upon patient dose modification rules 
from study A536-06).  All patients with treatment interruption will be initially treated with 
ACE-536 at a starting dose level of 0.8 mg/kg which has been determined to be safe and well 
tolerated by the SRT based on data from study A536-04.  Please refer to Table 2, Examples of 
Possible Starting Dose with Dose Modifications (Reductions and Titrations), for additional 
information regarding treatments administered. 
10.6. 
Safety Evaluation  
Safety will be evaluated by the 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 periodically 
throughout the trial, including AEs, SAEs, laboratory results (including hematology and 
chemistry), vital signs, and erythroid response data. 




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Observation on Dosing Day 
Action 
ACE-536 Dose Modification 
Hemoglobin ≥ 13 g/dL 
Hold dose and monitor 
patient weekly until 
hemoglobin < 12 g/dL 
Resume dosing once hemoglobin 
< 12 g/dL at the same dose level 
Hemoglobin increase ≥ 2 g/dL 
from the previous dosing day 
(not attributable to RBC 
transfusion) 
Continue dosing 
Reduce the dose by 1 dose level 
Non-hematologic AE ≥ Grade 3, 
or hematologic AE ≥ 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 the dose by 1 dose levela 
Grade 3 leukocytosis (WBC 
>100,000/μL) 
 
Discontinue treatment 
a Patients who require more than 2 dose reductions due to an AE should be discontinued from treatment and 
complete the EOT, PTFU, LTFU, and EOS visits. 
10.9. 
Individual Dose Titration 
10.9.1. 
Individual Dose Titration for NTD Patients 
For all NTD patients (with and without treatment interruption), the dose level will be assessed 
for titration at Day 1 of every cycle starting at C3D1 (e.g., C3D1, C4D1, C5D1, etc.).  The dose 
level should be titrated individually for each patient not to exceed the maximum dose level 
evaluated in the base study A536-04 as follows:   
 If hemoglobin increase from baseline is < 1.5 g/dL throughout the previous two 
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 two 
consecutive measurements during the previous two 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 two consecutive 
measurements during the previous two 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 two or more measurements 
performed during the screening period for base study A536-04 (for patients without treatment 
interruption) or study A536-06 (for patients with treatment interruption).  Hemoglobin 
measurements within 2 weeks following RBC transfusion will be excluded from both the 
calculation of the mean baseline hemoglobin and for evaluation of dose titration for a patient 
during treatment.  
10.9.2. 
Individual Dose Titration for TD Patients 
For all TD patients (with and without treatment interruption), the dose level will be assessed for 
titration at Day 1 of every cycle starting at C3D1 (e.g., C3D1, C4D1, C5D1, etc.).  The dose 


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level should be titrated individually for each patient not to exceed the maximum dose level 
evaluated in the base study A536-04 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). 
10.10. 
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 or interruption, the investigator 
should consult with the medical monitor to decide whether to continue dosing at the same dose 
level, reduce the dose level, delay the patient’s dose, or discontinue the patient’s treatment with 
ACE-536. 
Patient treatment interruption for site administrative reasons will be allowed between Cycle 17 
and 18 and between Cycle 35 and 36 upon discussion with the sponsor. 


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11. 
STUDY PROCEDURES  
Please refer to Section 2, Schedule of Events 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 
11.2.1. 
Local Laboratory Testing 
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, nucleated red blood cells (nRBCs), white blood cell (WBC) with 
differential, hemoglobin, hematocrit, reticulocyte count, platelet count, mean 
corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular 
hemoglobin concentration (MCHC), and red blood cell distribution width (RDW).   
 Serum chemistry: Sodium, potassium, 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. 
 Serum folate: required at screening for patients with treatment interruption to confirm 
eligibility; required at C1D1 and as required per schedule of events for all patients 
11.2.2. 
Central Laboratory Testing 
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. 
 nRBCs  
 EPO levels 
 Peripheral blood smear 
 Serum iron studies 
 Urine chemistries include but are not limited to: microalbumin and creatinine 


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11.3. 
Other Safety Assessments   
 Physical examination (including optional evaluation of gonadal size in males at 
specified timepoints) 
 Vital signs: weight, heart rate, systolic and diastolic blood pressure.   
 12-lead ECG  
 ADA testing: (Blood samples should be drawn and processed on-site for serum 
collection at the time points specified according to the relevant Laboratory Manual) 
 The occurrence of a new malignancy or premalignant lesion will be monitored as an 
event of interest and should be included as part of the assessment of adverse events 
throughout the course of the study.  Investigators are to report the development of any 
new malignancy or premalignant lesion as a serious adverse event, regardless of 
causal relationship to study drug, occurring at any time for the duration of the study, 
from the time of signing the ICF up to and including 3 years of follow up. 
11.4. 
Pharmacokinetic and Pharmacodynamic Assessments 
11.4.1. 
Pharmacokinetic Assessments 
PK assessment of ACE-536 concentrations will be performed periodically as outlined in 
Section 2, Schedule of Events.  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 relevant Laboratory Manual.   
11.4.2. 
Pharmacodynamic Evaluations 
PD assessments including hematologic laboratory assessments and transfusion information will 
be used to determine erythroid response to ACE-536.  Please note specific timepoints according 
to the Schedule of Events (Section 2): 
 An MRI will be performed at selected sites to evaluate LIC.  
 An MRI will be performed to evaluate EMH masses (unless site does not have the 
feasibility to perform the assessment). 
 A DXA will be performed at selected sites to evaluate total body, lumbar spine and 
total hip BMD. 
 Patients with leg ulcers should have regular assessment of the leg ulcer(s) throughout 
the study.  Photographs of the leg ulcer(s) pre- and post- dose should be provided 
when available to document any changes in the ulcer.   
 A 6MWT will be performed at selected sites for NTD patients. 
 Quality of Life tools including by not limited to the FACT-An and SF-36 
questionnaires will be assessed. 
 Biomarkers for iron metabolism may include serum iron, TIBC, transferrin, soluble 
transferrin receptor, calculated transferrin saturation, ferritin, and NTBI. 


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 Biomarkers related to the TGF superfamily and/or iron metabolism, such as 
hepcidin, GDF15, GDF8, GDF11, Activin A and others may be tested in blood 
samples, to be determined. 
 Hemoglobin electrophoresis samples will be collected for central analysis. 
 The sponsor may perform additional sample analysis for biomarkers for exploratory 
research purposes only. 


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12. 
STUDY SCHEDULE 
Please refer to Section 2, Schedule of Events 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 during the 28-day screening period for all 
patients. 
 Other than informed consent, procedures listed as part of the 28-day screening period 
are only applicable to patients with treatment interruption.   
 ECHO or MUGA (only required for patients with treatment interruption) can be 
performed up to 56 days prior to C1D1.  If performed as part of standard of care, 
ECHO/MUGA does not need to be repeated.   
 Patients with treatment interruption (defined as patients who complete the EOS visit 
for study A536-04 and are ≥ 28 days post EOS visit) will need to qualify per the 
additional inclusion criteria listed in Section 9.3.2.  
 Transfusion history will be collected from the EOS visit in study A536-04 through 
the C1D1 visit of A536-06 as available; TD patients 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.  For TD 
patients without treatment interruption, the baseline pre-transfusion hemoglobin 
threshold will be the mean of all documented pre-transfusion hemoglobin values 
during the 12 weeks prior to C1D1 of base study A536-04 or study A536-06 (for TD 
patients with treatment interruption).   
 Pregnancy test (urine or serum) and menstrual history is required of female patients 
of child bearing potential only. 
 Medical history for patients with treatment interruption will include medical events 
occurring after EOS visit in study A536-04 and prior to C1D1 for study A536-06.  
Medical history for patients without treatment interruption will be taken from study 
A536-04. 
 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. 
 Screen failure information will be maintained to document specific information, 
including but not limited to, reason for failure. 
12.2. 
Dosing Days and Interim Visits 
 Study procedures must be done prior to administration of study drug.  All patients 
must be assessed for eligibility prior to dosing on C1D1.  C1D1 MRI for LIC 


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(at selected sites) and EMH masses (unless site does not have feasibility to perform 
the assessment) and MRI of the spleen (if no prior splenectomy) can be performed up 
to 56 days prior to C1D1.  If performed as part of standard of care, MRI for LIC, MRI 
for EMH and spleen do not need to be repeated.  All screening and Cycle 1 Day 1 
procedure results required to confirm eligibility must be obtained and reviewed prior 
to study drug administration in A536-06.   
 On dosing days: Note that the patient dose must be calculated based on the patient’s 
weight on the day of dosing.  Starting dose level for C1D1, dose modification rules 
and titration rules must be reviewed and implemented prior to dosing as required per 
protocol (see Sections 10.7, 10.8, 10.9).  If a dose delay is required per the dose 
modification rules (Section 10.8), the patient will not be dosed.  The patient will 
return weekly for assessment of hematology results and AEs until the patient is 
eligible to receive the next dose of ACE-536 and start the next cycle. The patient 
should resume the study at the planned dosing cycle (e.g. if the patient missed a dose 
at C4D1, then they would resume dosing at C4D1 and not skip to C5D1). 
 The hematology results can be collected up to 24 hours prior to the dosing day.  For 
patients with treatment interruption, the hemoglobin should be available to confirm 
eligibility within one day prior to C1D1.  
 Patients should be monitored for AEs throughout the study. 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 should include an optional evaluation of gonadal size in male patients 
at timepoints specified in the Schedule of Events (Section 2). 
 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. 
 Patients with clinical signs of a change in spleen size or abnormality should have an 
abdominal ultrasound or MRI performed as needed throughout the study.   
 During treatment, if the pre-transfusion hemoglobin level 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). 
 Cycles 6 through 17:  Procedures and visits for Cycles 6 through 9, 10 through 13, 
and 14 through 17 follow the same procedures and visits as scheduled for Cycles 2 
through 5, with some exceptions as noted in the Schedule of Events (Section 2). 


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 Cycles 24 through 87: Patients may not exceed 87 cycles or 1825 days after first dose, 
whichever occurs first.  The last dose of ACE-536 may not be administered after Day 
1825, regardless of the number of cycles completed.  Procedures and visits for Cycles 
24 through 27, 28 through 31, 32 through 35, 36 through 39, 40 through 43, 44 
through 47, 48 through 51, 52 through 55, 56 through 59, 60 through 63, 64 through 
67, 68 through 71, 72 through 75, 76 through 79, 80 through 83 and 84 through 87  
follow the same procedures and visits as scheduled for Cycles 20 through 23, with 
some exceptions as noted in the Schedule of Events (Section 2). 
12.3. 
End of Treatment Visit 
 The EOT visit should occur 28 days (± 7 days) after the last dose of ACE-536.  
 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. 
 Patients who discontinue treatment early should complete the EOT visit at the time of 
discontinuation. 
12.4. 
Post-Treatment Follow-Up Visit 
 The PTFU visit should occur 2 months (± 7 days) after the last dose of ACE-536.  
 If a patient has a positive 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. 
12.5. 
Long-Term Follow-Up Visits 
 LTFU visits should occur beginning every 6 months (± 14 days) after the last dose of 
ACE-536 for 3 years after the last dose of ACE-536.  
12.6. 
End of Study Visit 
 The EOS visit should occur approximately 3 years after the last dose of ACE-536.  
 Physical exam should include an optional evaluation of gonadal size of male patients. 
12.7. 
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 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 will be provided as either a sterile liquid formulation or a lyophilized 
powder formulation for reconstitution with water.  Each single-use vial of the liquid formulation 
contains 25 mg of ACE-536 in a 0.5 mL Tris-buffered saline solution (50 mg/mL).  Each single-
use vial of the lyophilic formulation may contain either 25 mg, 50 mg, or 75 mg ACE-536 for 
reconstitution with 0.5 mL, 1 mL, or 1.5 mL water, respectively, to form a citrate-buffered 
solution (50 mg/mL) for injection. 
13.3. 
Study Drug Storage 
ACE-536 liquid formulation is stored frozen at  -65°C until use. 
ACE-536 lyophilic formulation is stored at 2-8°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 4 injections will be 
administered per dose.   
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 product composition, drug handling, administration, storage, and disposal instructions.  


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14. 
ASSESSMENT OF SAFETY 
14.1. 
Adverse Event Definitions 
Adverse Event 
An 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, 
Electrocardiogram (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 IB. 
Events Not to Be Considered as Adverse Events 
Pre-existing medical conditions/signs/symptoms present before the screening period (from study 
A536-04 for patients without treatment interruption; from study A536-06 for patients with 
treatment interruption) 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 AEs. 
Serious Adverse Event 
An 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. 
 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 


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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.  
Adverse Events of Special Interest 
Events of new malignancy and premalignant lesions (excluding benign tumors or benign 
neoplasia) are to be reported as described in Section 14.7 within 24 hours of the investigator’s 
knowledge of the event by fax, or other appropriate method, using the SAE Report Form, and 
must be considered an “Important Medical Event” even if no other serious criteria apply; these 
events must also be documented in the appropriate page(s) of the CRF and subject’s source 
documents.  Documentation of the diagnosed malignancy must be provided at the time of 
reporting as a serious adverse event (e.g., any confirmatory histology or cytology results, X-rays, 
CT scans, etc.). 
Malignancies or cancerous tumors are lesions capable of invading into adjacent tissues, and may 
be capable of spreading to distant tissues.  A benign tumor has none of those properties. 
Malignancy or cancer is characterized by anaplasia, invasiveness, and metastasis.   
Premalignant or precancerous lesions refer to a state of disordered morphology of cells that is 
associated with an increased risk of cancer.  If left untreated, these conditions may lead to 
cancer.  Such conditions are usually either dysplasia or benign neoplasia (and the dividing line 
between those is sometimes blurry).  Sometimes the term "precancer" is used to describe 
carcinoma in situ, which is a noninvasive cancer that has not progressed to an aggressive, 
invasive stage.  Not all carcinoma in situ will progress to invasive disease. 
Premalignant lesions are morphologically atypical tissue which appears abnormal under 
microscopic examination and in which cancer is more likely to occur than in its apparently 
normal counterpart. 
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. 


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14.3. 
Severity 
Investigators must evaluate the severity/intensity of AEs according to the current active minor 
version of the NCI-CTCAE v4.0, current minor version, preferentially using the graded scales.  
If there is a change in severity of an AE, 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 
(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 AEs 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 patients or observed during or following the 
course of investigational product administration on the appropriate CRF page.  For patients 


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without treatment interruption, any AE ongoing at the time of EOT in A536-04 needs to be 
re-entered as an AE in A536-06.  For patients with treatment interruption, all non-serious AEs 
occurring after signing of the ICF until a patient is dosed on C1D1 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 (EOS 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 
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 (EOS 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. 


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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, 
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. 
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 ICH and 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 


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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 
Analysis Populations:  
The modified Intent-to-Treat (mITT) Population: All patients enrolled in the study who 
received at least one dose of ACE-536.  
Efficacy Evaluable (EE) Population: All patients administered at least one dose of ACE-536 
and have 1) at least three hemoglobin assessments over an 8-week period, not influenced by 
transfusion, in NTD patients, OR, 2) at least 8 consecutive weeks of transfusion frequency data 
in TD patients. 
Pharmacokinetics Population: All patients who received at least 1 dose of ACE-536 during 
Study A536-06 and have sufficient serum ACE-536 values 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, SD, median, and range).   
15.2.2. 
Primary Endpoint Analysis  
To assess clinical safety, all AEs, vital sign measurements and clinical laboratory information 
will be summarized.  AEs 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. 
Disposition rates will be summarized. 
15.2.3. 
Secondary Analysis 
All efficacy analyses will be performed for the EE and mITT populations. 
Efficacy will be evaluated by determining the proportion of β-thalassemia patients who have an 
erythroid response, defined as 1) a mean hemoglobin increase ≥ 1.0 g/dL or ≥ 1.5 g/dL over an 
8-week or 12-week period as compared to baseline, not influenced by RBC transfusion in NTD 
patients or 2) ≥ 20% or ≥ 50% reduction in RBC transfusion burden over an 8-week or 12-week 
period as compared to the 8-week or 12-week period pretreatment in TD patients. 
Baseline hemoglobin for NTD patients will be the average of two or more measurements 
performed during the screening period for base study A536-04 (for patients without treatment 


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interruption) or study A536-06 (for patients with treatment interruption).  Hemoglobin 
measurements within 2 weeks following RBC transfusion will be excluded. 
Transfusion burden for TD patients will be defined as the ratio of RBC transfusion units 
(or mLs) transfused during an interval divided by the duration 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 8 or 12 weeks prior to Cycle 1 Day 1 of 
base study A536-04 (will also be analyzed using pretreatment period of study A536-06 for 
patients with treatment interruption).  An interval during the treatment plus follow-up period will 
be defined as an 8-week or 12-week interval after Cycle 1 Day 1 of study A536-06. 
The erythroid response, as defined above, will also be evaluated using other pre-treatment and 
on-treatment intervals and other % reductions in RBC units transfused, including 100% 
(transfusion-free). 
The erythroid response will be summarized using both a point estimate and its exact 95% CI 
based on binomial distribution. 
No direct comparison testing with concurrent or historical controls will be performed.   
Analysis of other secondary efficacy/PD endpoints will include: 
 Time to erythroid response and duration of erythroid response 
 Change/percent change from pretreatment in mean transfusion burden in TD patients 
 Change in mean hemoglobin level in NTD patients 
 Change in pre-transfusion hemoglobin levels in TD patients (including analysis of 
patient subset with < 20% change in transfusion burden)   
 Change from baseline in erythropoiesis parameters, including EPO, reticulocytes, 
nucleated RBCs, and soluble transferrin receptor 
 Change from baseline in hemolysis parameters, including total/indirect bilirubin, and 
LDH  
 Change from baseline in iron overload and iron metabolism parameters, including 
serum iron, total iron binding capacity (TIBC), transferrin, calculated transferrin 
saturation, soluble transferrin receptor, ferritin, non-transferrin bound iron (NTBI), 
hepcidin, iron chelation therapy use and LIC by MRI 
The exploratory endpoints will include evaluation of biomarkers related to the TGF- 
superfamily, quality of life, change in EMH mass size, change in spleen size, change in BMD, 
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 


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analyzed using Kaplan-Meier method to estimate the survival curve and median time to event 
and 95% CI. 
15.2.4. 
Pharmacokinetics Analysis  
PK parameters for ACE-536, such as maximum serum concentration (Cmax), time to maximum 
concentration (tmax), and area under the concentration/time curve (AUC), will be estimated using 
non-compartmental and/or single compartment analysis, as appropriate.  Dose proportionality 
may be assessed using the exposure data (e.g., Cmax, AUC) 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, as 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 ADA on ACE-536 PK and drug exposure if ADA tests are deemed positive. 
15.3. 
Determination of Sample Size 
There is no formal sample size calculation for the study although up to 72 patients may 
participate from the A536-04 study.   
15.4. 
Interim Analysis 
Safety and erythroid response data will be reviewed periodically throughout the study. A formal 
interim analysis of the safety and efficacy data will be performed to support registration filings. 
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. 


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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 the United States Food and Drug Administration (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 (i.e., 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 U.S. Law.  This Web site will not include information that can identify patients, but 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). 


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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 
CRFs 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. Musallam KM, Rivella S, Vichinsky E, Rachmilewitz EA. Non-transfusion-dependent 
thalassemias. Haematologica 2013;98:833-44. 
7. Weatherall DJ. The inherited diseases of hemoglobin are an emerging global health burden. 
Blood 2010;115:4331-6. 


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23. 
APPENDICES 
23.1. 
Appendix 1: National Cancer Institute (NCI) Common Terminology 
Criteria for Adverse Events (CTCAE)  
See http://evs.nci.nih.gov/ftp1/CTCAE 

SAP_001.pdf: 
Official Protocol Title: 
NCT number: 
Document Date: 
An Open-Label Extension Study to Evaluate the Long-Term 
Effects of ACE-536 in Patients with β-Thalassemia Previously 
Enrolled in Study A536-04
10-Jun-2020
NCT02268409


 
 
 
Statistical Analysis Plan 
 
 
An Open-Label Extension Study to Evaluate the 
Long-Term Effects of ACE-536 in Patients with 
β-Thalassemia Previously Enrolled in Study A536-04 
Sponsor: 
Acceleron Pharma Inc. 
128 Sidney Street 
Cambridge, MA 02139, USA 
Tel: 
 
Fax: 
 
Investigational Product: 
Luspatercept (ACE-536) 
Protocol Number: 
A536-06 
EudraCT Number: 
2014-001281-94 
Version Number: 
2 (Final) 
Date: 
10 June 2020 
 
 
 
 
 
 
CONFIDENTIALITY STATEMENT 
The information contained in this document, especially unpublished data, is the property of 
Acceleron Pharma Inc. (or under its control), and therefore provided to you in confidence for 
review by you and/or your staff. It is understood that this information will not be disclosed to 
others without written authorization from Acceleron. 
 
 
PPD
PPD


P


A536-06 
 
Statistical Analysis Plan 
Confidential and Proprietary  
3 
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TABLE OF CONTENTS 
Table of Contents .............................................................................................................................3 
1. 
Introduction ...................................................................................................................5 
2. 
Study Objectives ...........................................................................................................5 
3. 
Overall Study Design ....................................................................................................6 
3.1. 
Study Design .................................................................................................................6 
3.2. 
Treatment Discontinuation ...........................................................................................8 
3.3. 
Sample Size ..................................................................................................................9 
4. 
Analysis Populations ....................................................................................................9 
4.1. 
The Intent-To-Treat (ITT) Population ..........................................................................9 
4.2. 
Safety Population ..........................................................................................................9 
4.3. 
Pharmacokinetics (PK) Population ...............................................................................9 
5. 
Statistical Methodology ................................................................................................9 
5.1. 
Definitions ....................................................................................................................9 
5.2. 
General Considerations ...............................................................................................11 
5.3. 
Disposition of Patients ................................................................................................11 
5.4. 
Demographic, Baseline Characteristics, and Disease History ....................................11 
5.5. 
Study Drug Exposure ..................................................................................................12 
5.6. 
Prior and Concomitant Medication and Procedures ...................................................12 
5.6.1. 
Prior and Concomitant Medication .............................................................................12 
5.6.2. 
RBC Transfusions .......................................................................................................12 
5.6.3. 
Non-Medication Procedures .......................................................................................12 
5.7. 
Efficacy Analyses .......................................................................................................13 
5.7.1. 
Primary Efficacy Endpoint .........................................................................................13 
5.7.2. 
Secondary Efficacy Endpoints ....................................................................................13 
5.7.2.1. 
Erythroid Response .....................................................................................................13 
5.7.2.2. 
Time to and Duration of Erythroid Response .............................................................14 
5.7.2.3. 
Change of Hemoglobin Level and Transfusion Burden .............................................15 
5.7.2.4. 
Iron-Related Parameter Analysis ................................................................................15 
5.7.2.5. 
Erythropoiesis Parameters ..........................................................................................16 
5.7.2.6. 
Hemolysis Parameters ................................................................................................16 
5.7.2.7. 
Liver Iron Content (LIC) ............................................................................................16 


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Statistical Analysis Plan 
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5.7.3. 
Exploratory Endpoints ................................................................................................17 
5.7.3.1. 
Biomarkers Related to TGF- ....................................................................................17 
5.7.3.2. 
Quality of Life (QoL) Questionnaire ..........................................................................17 
5.7.3.3. 
Other Analysis. ...........................................................................................................17 
5.7.4. 
Subgroup Analysis ......................................................................................................17 
5.8. 
Safety Analysis ...........................................................................................................18 
5.8.1. 
Adverse Events ...........................................................................................................18 
5.8.2. 
Laboratory Evaluations ...............................................................................................19 
5.8.3. 
Vital Signs ..................................................................................................................19 
5.8.4. 
Echocardiogram (ECG) Results .................................................................................19 
5.8.5. 
Physical Examination .................................................................................................20 
5.8.6. 
Post-treatment Follow-up and Long-term Follow-up .................................................20 
5.9. 
Pharmacokinetics Analysis .........................................................................................20 
5.9.1. 
Pharmacokinetic Sampling Schedule .........................................................................20 
5.9.2. 
Data Handling .............................................................................................................20 
5.9.3. 
Pharmacokinetic Analysis ..........................................................................................21 
5.10. 
Interim Analysis ..........................................................................................................21 
5.11. 
Protocol Deviations ....................................................................................................21 
5.12. 
Data Handling .............................................................................................................21 
5.12.1. 
Analysis Visit Window ...............................................................................................21 
5.12.2. 
Handling of Missing Data ...........................................................................................22 
5.12.3. 
Missing Dates for Adverse Event ...............................................................................22 
5.12.4. 
Missing Dates for Concomitant Medication ...............................................................22 
5.12.5. 
Missing Dates for Disease Diagnosis Date .................................................................23 
5.12.6. 
Changes in Conduct or Planned Analyses from the Protocol .....................................23 
6. 
References ...................................................................................................................24 
7. 
Appendices .................................................................................................................25 
7.1. 
Appendix 1 - FACT-An and SF-36 Scoring Algorithm .............................................25 
7.2. 
Appendix 2 - List of Abbreviations ............................................................................30 
7.3. 
Appendix 3 - Schedule of Events for Study A536-04 ................................................33 
7.4. 
Appendix 4 - Schedule of Events for Study A536-06 ................................................36 
 
 


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Statistical Analysis Plan 
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1. 
INTRODUCTION 
This statistical analysis plan (SAP) describes the statistical methods to be used for the analysis of 
Acceleron Protocol A536-06.  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 05 dated 12 September 2017 and eCRF v7 dated 14 March 2018.  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 long-term safety and tolerability of ACE-536 in patients with 
β-thalassemia who were previously enrolled in study A536-04.  
Secondary Objectives: 
 To evaluate erythroid response defined as the proportion of patients with:  
 Mean hemoglobin increase ≥ 1.5 g/dL over a continuous 12-week interval 
compared to baseline in non-transfusion dependent (NTD) patients, not 
influenced by red blood cell (RBC) transfusion, OR  
 Reduction in RBC transfusion burden by ≥ 50% over a continuous 12-week 
interval compared to the 12 weeks prior to the start of treatment in 
transfusion dependent (TD) patients.  
 To evaluate erythroid response defined as proportion of patients with:  
 Mean hemoglobin increase ≥ 1.5 g/dL over a continuous 8-week interval 
compared to baseline in NTD patients, not influenced by RBC transfusion, 
OR  
 Reduction in RBC transfusion burden by ≥ 20% over a continuous 8-week 
interval compared to the 8 weeks prior to the start of treatment in TD 
patients.  
 To evaluate the time to erythroid response and duration of erythroid response.  
 To evaluate the mean change from baseline over an 8- or 12-week period in hemoglobin 
level in NTD patients not influenced by RBC transfusion  
 To evaluate the mean % change from baseline in transfusion burden over an 8- or 
12-week period in TD patients.  
 To evaluate the mean change in pre-transfusion hemoglobin levels in TD patients  
 To evaluate changes in markers of erythropoiesis, hemolysis, iron overload, and iron 
metabolism  
 To examine the pharmacokinetic (PK) profile of ACE-536 in patients with 
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Exploratory Objectives:  
 To evaluate biomarkers related to the transforming growth factor beta (TGF-β) 
superfamily  
 To evaluate patient self-reported quality of life using tools including but not limited 
to the Functional Assessment of Cancer Therapy-Anemia Scale (FACT-An) and 
Short Form (36) Health Survey (SF-36) questionnaires  
 To evaluate change in extramedullary hematopoiesis (EMH) mass size by MRI  
 To evaluate change in spleen size by MRI  
 To evaluate change in bone mineral density (BMD) by DXA  
 To evaluate change in leg ulcers  
 To evaluate change in the 6-minute walk test (6MWT) distance in NTD patients  
3. 
OVERALL STUDY DESIGN 
This open-label extension study will evaluate the effects of up to 60 months of ACE-536 
treatment in patients with β-thalassemia previously enrolled and treated with ACE-536 for up to 
3 months in study A536-04.  The base study (A536-04) is a phase 2, open-label, ascending dose 
study to evaluate the effects of ACE-536 in patients with β-thalassemia.  A total of up to 
64 patients may be enrolled in study A536-04 and may be eligible for study A536-06. 
3.1. 
Study Design 
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. For these patients, Cycle 1 Day 1 
(C1D1) of study A536-06 may take place 28 (± 7) days after the last dose administered in study 
A536-04, which may coincide with the patient’s A536-04 End of Treatment (EOT) visit.  These 
patients are considered “patients without treatment interruption”.  
Patients who complete the EOS visit for study A536-04 and are ≥ 28 days post EOS visit are 
considered “patients with treatment interruption” and will be re-assessed for eligibility by 
meeting all eligibility criteria plus additional inclusion criteria, as defined in Section 9.3 of the 
protocol.  
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 be treated as 
patients with treatment interruption and should not begin study A536-06 C1D1 until they have 
reached ≥ 28 days post EOS visit from study A536-04 so that new baseline assessments can be 
measured. 
For patients without treatment interruption, transfusion status (NTD or TD) will carry-over from 
the base study A536-04.  For NTD patients with treatment interruption, transfusion status will be 
reassessed prior to C1D1 of study A536-06.  NTD patients with treatment interruption are 
defined as patients who require transfusion of < 4 units of RBCs over the 8 weeks prior to Cycle 




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Patients will participate in the extension study A536-06 for up to 97 months, including a 28-day 
(1 month) screening period, a 60-month treatment period and a 3-year (36 months) follow-up 
period.  
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 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.   
 


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3.3. 
Sample Size 
There is no formal sample size calculation for the study although up to 64 patients may participate 
from the A536-04 study. 
4. 
ANALYSIS POPULATIONS 
4.1. 
The Intent-To-Treat (ITT) Population 
The intent-to-treat (ITT) population consists of all patients enrolled in the study who received at 
least one dose of ACE-536 in the A536-06 study.  This population will be used for all efficacy 
analysis.   
For patients who directly rolled over from A536-04 study, both A536-04 study and A536-06 
study data will be included for the analysis. 
For interrupted patients, only A536-06 study data will be included for the analysis. 
4.2. 
Safety Population 
Same as ITT population. 
4.3. 
Pharmacokinetics (PK) Population 
The pharmacokinetics population will include all patients who received at least 1 dose of 
ACE-536 during Study A536-06 and have sufficient serum ACE-536 values for PK analysis. 
5. 
STATISTICAL METHODOLOGY 
5.1. 
Definitions 
Transfusion Status 
For patients without treatment interruption, transfusion status (NTD or TD) will carry-over from 
the base study A536-04.  
For NTD patients with treatment interruption, transfusion status will be reassessed prior to C1D1 
of study A536-06. NTD patients with treatment interruption are defined as patients who require 
transfusion of < 4 units of RBCs over the 8 weeks prior to Cycle 1 Day 1 of study A536-06.  
For TD patients with treatment interruption, transfusion status will carry-over from the base 
study A536-04.  
Transfusion Dependent (TD) Defined in Base Study A536-04  
Based on the protocol, TD patients are defined as those who required ≥ 4 units of RBCs every 8 
weeks (confirmed over 6 months prior to study A536-04 Cycle 1 Day 1). For statistical 
programming purposes, a patient is defined as TD if the total RBCs is ≥ 11 units during 26 
weeks on or prior to study A536-04 Cycle 1 Day 1.  
 


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Non-transfusion Dependent (NTD) Defined in Base Study A536-04 
NTD patients are those who do not meet the above definition for TD.  For statistical 
programming purposes, NTD patients are defined as those who received < 11 units of RBCs 
during 26 weeks on or prior to study A536-04 Cycle 1 Day 1.  
Baseline  
For patients who roll over to Study A536-06 without interruption, the baseline for Study 
A536-04 will be used as the baseline for Study A536-06 for all measurements, as further defined 
below.  For patients who enter Study A536-06 with interruption, the baseline for Study A536-06 
will be used, as further defined below.  For assessments that are not scheduled to be done or with 
an inadequate data collection window at the beginning of Study A536-06, the baseline for Study 
A536-04 will be used as the baseline for Study A536-06. 
Baseline Hemoglobin  
Baseline hemoglobin will be the 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 prior to 
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   
A transfusion amount reported in “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.   
Post-Treatment Follow-Up (PTFU) 
The PTFU visit should occur 2 months (± 7 days) after the last dose of ACE-536.  If a patient has 
a positive 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. 
Long-Term Follow-Up (LTFU) 
LTFU visits should occur beginning every 6 months (± 14 days) after the last dose of ACE-536 
for 3 years after the last dose of ACE-536.  
 
 


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End of Study (EOS)   
Procedures and evaluations for the end of study visit should be performed approximately 3 years 
after the last dose of ACE-536. 
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 
provided.  Percentages will use column totals as the denominator unless otherwise indicated.  For 
time to event variables, the Kaplan-Meier 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. 
Data 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.  
5.4. 
Demographic, Baseline Characteristics, and Disease History 
The following baseline and demographic characteristics will be summarized by descriptive 
statistics for the ITT population as well as for TD and NTD patients separately:  
 Race, ethnicity, age, sex, height, weight  
 Splenectomy (yes or no), iron chelation therapy (yes or no), prior β-thalassemia 
treatment (yes or no) 
 Baseline hemoglobin for NTD patients only and baseline transfusion burden for TD 
patients only.  
 Baseline LIC and serum ferritin 
 Baseline EPO and time from diagnosis in years 
 Baseline iron intake 
Demographic and baseline data, medical history, and disease history data will be listed for each 
patient.  Medical History data will be summarized using descriptive statistics. 


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5.5. 
Study Drug Exposure 
Study drug exposure will be descriptively summarized 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 (titration).  
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 study drug in mg a patient received during the 
treatment period. 
Study drug administration details will be listed for each patient. 
5.6. 
Prior and Concomitant Medication and Procedures 
5.6.1. 
Prior and Concomitant Medication 
The prior and concomitant medications are coded with WHO dictionary.  The medications will 
be summarized for the safety 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 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 20.0.  All non-medication 
procedures will be listed for each patient. 


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5.7. 
Efficacy Analyses 
No formal hypothesis testing is planned.  All efficacy endpoint will be performed using the ITT 
population.  
In general, the 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 the efficacy analysis. 
 For each patient, all efficacy endpoints will be derived based on an analysis cutoff 
day, defined as the last dose + 56 days or the last date from transfusion record data, 
whichever is earlier.  
5.7.1. 
Primary Efficacy Endpoint 
Since the primary objective for this study is to evaluate the long-term safety and tolerability of 
ACE-536 in patients with β-thalassemia who were previously enrolled in study A536-04, there is 
no primary efficacy endpoint specified for this study. 
5.7.2. 
Secondary Efficacy Endpoints 
5.7.2.1. 
Erythroid Response 
Erythroid response rates will be defined as below for NTD and TD patients. 
For 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 
 The proportion of patients with a mean increase of ≥ 1.0 g/dL from pretreatment 
hemoglobin during any rolling 24 weeks 
 The proportion of patients with a mean increase of ≥ 1.5 g/dL from pretreatment 
hemoglobin during any rolling 24 weeks 
 The proportion of patients with a mean increase of ≥ 1.0 g/dL from pretreatment 
hemoglobin during weeks 13-24 
 The proportion of patients with a mean increase of ≥ 1.5 g/dL from pretreatment 
hemoglobin during weeks 13-24 
 The proportion of patients with a mean increase of ≥ 1.0 g/dL from pretreatment 
hemoglobin during weeks 37-48 


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 The proportion of patients with a mean increase of ≥ 1.5 g/dL from pretreatment 
hemoglobin during weeks 37-48 
For TD patients: 
 The proportion of patients who have ≥ 20% reduction in RBC transfusion burden 
compared to pretreatment using rolling 8-week intervals  
 The proportion of patients who have ≥ 33% reduction in RBC transfusion burden 
compared to pretreatment using rolling 8-week intervals 
 The proportion of patients who have ≥ 50% reduction in RBC transfusion burden 
compared to pretreatment using rolling 8-week intervals  
 The proportion of patients who have ≥ 20% reduction in RBC transfusion burden 
compared to pretreatment using rolling 12-week intervals  
 The proportion of patients who have ≥ 33% reduction in RBC transfusion burden 
compared to pretreatment using rolling 12-week intervals  
 The proportion of patients who have ≥ 50% reduction in RBC transfusion burden 
compared to pretreatment using rolling 12-week intervals  
 The proportion of patients who have ≥ 33% reduction in RBC transfusion burden 
compared to pretreatment during weeks 13-24  
 The proportion of patients who have ≥ 50% reduction in RBC transfusion burden 
compared to pretreatment during weeks 13-24  
 The proportion of patients who have ≥ 33% reduction in RBC transfusion burden 
compared to pretreatment using rolling 24-week intervals  
 The proportion of patients who have ≥ 50% reduction in RBC transfusion burden 
compared to pretreatment using rolling 24-week intervals  
 The proportion of patients who have ≥ 33% reduction in RBC transfusion burden 
compared to pretreatment during weeks 37-48  
 The proportion of patients who have ≥ 50% reduction in RBC transfusion burden 
compared to pretreatment during weeks 37-48  
 The proportion of patients who have no RBC transfusions ≥ 8 weeks 
For each of the above defined erythroid response rates, a point estimate will be presented along 
with the exact 95% confidence interval based on binomial distribution. 
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 


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 For NTD patients, mean increase of ≥ 1.5 g/dL from pretreatment hemoglobin during 
any rolling 12 weeks 
 For NTD patients, mean increase of ≥ 1.0 g/dL from pretreatment hemoglobin during 
any rolling 24 weeks 
 For NTD patients, mean increase of ≥ 1.5 g/dL from pretreatment hemoglobin during 
any rolling 24 weeks 
 For TD patients, ≥ 33% reduction in RBC transfusion burden compared to 
pretreatment using 12-week intervals 
 For TD patients, ≥ 50% reduction in RBC transfusion burden compared to 
pretreatment using 12-week intervals 
 For TD patients, ≥ 33% reduction in RBC transfusion burden compared to 
pretreatment using rolling 24-week intervals 
 For TD patients, ≥ 50% reduction in RBC transfusion burden compared to 
pretreatment using rolling 24-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- or 24-week interval achieving above erythroid response.  
Duration of erythroid response will be defined as the time from the starting date of the first 
rolling 12- or 24-week interval achieving response to the last date of the last consecutive rolling 
12- or 24-week interval achieving response.  When there are multiple disjointed 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. 
Erythroid responders may experience multiple periods of response (≥ 8 weeks, ≥ 12 weeks, or 
≥ 24 weeks).  For these responders, the number of episodes and cumulative duration will be 
summarized by NTD and TD separately.  As an exploratory analysis, the cumulative duration of 
the multiple responses will be analyzed using Kaplan-Meier method. 
5.7.2.3. 
Change of Hemoglobin Level and Transfusion Burden 
The mean change in hemoglobin level from baseline over 8- or 12-week period in NTD patients 
and the mean % change from baseline in transfusion burden over an 8- or 12-week period in TD 
patients will be summarized.  In addition, the mean change in pre-transfusion hemoglobin levels 
in TD patients (including analysis of patient subset with < 20% change in transfusion burden) will 
also be analyzed.  Summary statistics will be presented for these parameters.  
5.7.2.4. 
Iron-Related Parameter Analysis 
Selected iron metabolism parameters will be taken at Day 1 of Cycles 1, 5, 9, 13, 17, 21, Day 1 
of every 4 subsequent cycles until cycle 85, EOT, and EOS visits.  Descriptive statistics of actual 


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values, absolute and percentage change from baseline values at each post-baseline time point will 
be presented.  A shift table will be produced for ferritin from baseline to post-baseline.   
Plots of iron parameters (serum iron, TIBC, transferrin, calculated transferrin saturation, ferritin, 
NTBI) for both mean observed values and mean absolute change from baseline over time will be 
presented. 
5.7.2.5. 
Erythropoiesis Parameters 
Erythropoiesis parameters include serum EPO levels, reticulocytes, nucleated RBCs, soluble 
transferrin receptor, hemoglobin electrophoresis, and folate.   
EPO will be taken at Day 1 of Cycle 1, Days 1 and 8 of Cycles 5, 9, 13, 17, Day 1 of every 4 
subsequent cycles until cycle 85, EOT, and EOS.  Reticulocytes and nucleated RBCs will be 
taken at Screening, Days 1 and 8 of Cycles 1, 3,5,7,9,11,13,15,17, Day 1 of Cycles, 
2,4,8,10,12,14,16 and Cycles 18 to 87, EOT and EOS visits.  Soluble transferrin receptor will be 
taken at Day 1 of Cycles 1, 5, 9, 13, 17, Day 1 of every 4 subsequent cycles until Cycle 85, EOT, 
and EOS.  Hemoglobin electrophoresis will be taken at Day 1 of Cycles 1, 5, 13, 21, 29, 37, 45, 
53, 61, 69, 77, EOT, and EOS. Serum folate will be taken at screening for patients with treatment 
interruption to confirm eligibility, and required at Day 1 of Cycles 1, 5, 13, 21, 29 37, 45, 53, 61, 
69, 77, and EOT. 
Descriptive statistics of observed values, absolute and percentage change from baseline values at 
each post-baseline visit will be presented for all patients.  Plots of erythropoiesis parameters for 
mean observed values, mean change, and mean percentage change from baseline over time will 
be presented.  
5.7.2.6. 
Hemolysis Parameters 
Hemolysis parameters include total bilirubin, indirect bilirubin, and lactate dehydrogenase 
(LDH). Measurements will be done at Screening, Day 1 of Cycles 1, 3, 5, 9, 13, 27, 2, 25, 29, 33, 
37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, EOT, and EOS visits.  Descriptive statistics of 
observed values, absolute and percentage change from baseline values at each post-baseline visit 
will be presented.  Plots of hemolysis parameters for mean observed values, mean change, and 
mean percentage change from baseline over time will be presented. 
5.7.2.7. 
Liver Iron Content (LIC) 
MRI for liver iron content (LIC) will be performed.  Descriptive statistics of actual values, absolute 
and percentage change from baseline at post-baseline visits will be presented.  The summary will 
be done by NTD/TD, baseline LIC ≥ 3 and < 3 mg/g dw subgroups and baseline iron chelation 
therapy (ICT) use (yes vs. no) respectively.  
LIC responses will be defined as LIC reduction ≥ 1 mg/g dw at post-baseline visits compared 
with baseline.  LIC response rates along with the exact 95% confidence interval will be estimated 
for NTD and TD patients respectively, as well as by baseline LIC (< or > = 3 mg/g dw) and 
baseline ICT use (yes vs. no).  As an exploratory analysis, LIC change from baseline will be 
summarized by TD responders and non-responders, where TD responder is defined as 33% 
reduction in rolling 24 week and 50% reduction in rolling 12 weeks, respectively.  


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5.7.3. 
Exploratory Endpoints 
5.7.3.1. 
Biomarkers Related to TGF-  
Biomarkers hepcidin, GDF8, GDF11, and GDF15 will be presented in the listing.  
5.7.3.2. 
Quality of Life (QoL) Questionnaire 
QoL questionnaires including FACT-An and SF-36 will be completed at Day 1 of Cycles 1, 5, 9, 
13, 17, 25, 33, 41, 49, 57, 65, 73, 81, and EOT.  The scoring algorithms for FACT-An and SF-36 
are included in Appendix 1. 
Individual subscale scores and total scores will be summarized by visit for FACT-An, FACIT-F, 
and SF-36.  FACIT-F scores will also be summarized by visit for TD and NTD groups respectively. 
NTD-PRO score will be listed and summarized by visit for NTD group only.  All derived subscale 
and total scores along with all individual responses will be listed for each patient.  
Pairwise correlations among 6MWT, FACIT-F, SF-36, and hemoglobin will be evaluated at Week 
16 and 48. In addition, correlation between NTD-PRO Tiredness and Weakness domain score and 
FACIT-F score will be evaluated for the during of study. 
5.7.3.3. 
Other Analysis. 
The following additional exploratory endpoints will be presented in listings only:  
 Extramedullary hematopoiesis (EMH) mass size by MRI  
 Spleen size by MRI (in subset of patients with no prior-splenectomy) 
 %change and absolute change of bone mineral density (BMD) raw score and z-score 
by DXA 
 Leg ulcer size (area) 
 Change and %change in 6-minute-walk test (6MWT) distance in NTD patients 
 Hormone parameters (including estradiol, free testosterone, and total testosterone) 
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 years) 
 Sex (male and female) 
 Prior iron chelation therapy (yes and no) 
 Baseline LIC (< 3, 3-5, ≥ 5 mg/g dw) 
 Baseline EPO (< 200 and ≥ 200 IU/L) 
 Splenectomy (yes and no) 
 Baseline iron intake (< 0.4 mg Fe/kg/day and ≥ 0.4 mg Fe/kg/day) 


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 Baseline transfusion burden (RBC transfusion > 10 units per 12 weeks vs < =10 units 
per 12 weeks) 
5.8. 
Safety Analysis 
The safety endpoints will be summarized using the Safety Population.  The safety endpoints 
include treatment emergent adverse events, changes in laboratory tests, vital signs and ECG’s. 
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. 
Adverse Events 
For patients with treatment interruption, all non-serious AEs occurring after signing of the ICF 
until a patient is dosed on C1D1 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 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 for all dosed: 
 Number and percentage of patients reporting each AE, categorized by System Organ 
Class (SOC) and Preferred Term (PT) 
 Number and percentage of patients reporting each AE experienced by ≥ 5% and 
≥ 10% of patients in all patients by PT 
 Number and percentage of patients reporting SAE, categorized by SOC and PT 
 Number and percentage of patients reporting Grade ≥ 3 AE, categorized by SOC and 
PT 
 Number and percentage of patients reporting related AE, categorized by SOC and PT 
 Number and percentage of patients reporting AE leading to drug withdrawal, 
categorized by SOC and PT 
 Number and percentage of AE of interests by cycle 
Note that counting will be by patient, not event, and patients are only counted once within each 
SOC or PT.  If a patient 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 study drug discontinuation; 
4) death. 
Concomitant medication use, severity, time to the worst CTCAE grade and recovery will be 
listed and summarized. 


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5.8.2. 
Laboratory Evaluations 
The following laboratory parameters will be measured over time:   
Hematology:  
RBC, white blood cell (WBC) with differential, hemoglobin, hematocrit, haptoglobin (optional), 
reticulocyte count, platelet count, mean corpuscular volume (MCV), mean corpuscular 
hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell 
distribution width (RDW), and nucleated RBCs (nRBCs).   
Chemistry:  
Sodium, potassium, chloride, carbon dioxide/bicarbonate (optional), aspartate aminotransferase 
(AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total bilirubin, indirect 
bilirubin, alkaline phosphatase, blood urea nitrogen (BUN)/urea, creatinine, gamma-glutamyl 
transpeptidase (GGT), calcium, phosphorus, glucose, amylase, lipase, total protein, albumin, and 
uric acid. 
Urine Analysis:  
Local lab dipstick analysis will be done for specific gravity, protein, glucose, ketones, blood, 
leukocyte esterase, and nitrite, with microscopic examination if performed.  
Urine Chemistry:  
Microalbumin and creatinine will be performed by the central lab; albumin/creatinine ratio will 
be provided. 
Categorical and numeric variables will be presented separately.  Actual values and changes in 
hematology and biochemistry laboratory values from baseline will be summarized by time point. 
Shift tables for the hematology 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 listed. 
Peripheral blood smears data will be listed only.  All laboratory values will be listed for all 
patients. 
5.8.3. 
Vital Signs 
Vital signs parameters include weight (kg), heart rate, systolic and diastolic blood pressure, 
respiratory rate, and temperature (◦C).  For each parameter at each time point, the change from 
baseline to post baseline will be summarized.  
Plots of vital sign parameters for both mean observed and mean change from baseline will be 
presented for each parameter.  Vital signs will also be listed for all patients. 
5.8.4. 
Echocardiogram (ECG) Results 
The quantitative ECG assessments (ventricular rate, QRS width, PR interval, and QTc interval) 
will be summarized at each time point. 


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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]. 
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 patient, then an ‘Unknown’ category will be presented. 
ECG results will be listed for all patients. 
5.8.5. 
Physical Examination 
Physical exam details will be listed only. 
5.8.6. 
Post-treatment Follow-up and Long-term Follow-up 
A listing of post-treatment follow-up and Long-term follow-up data including the visit date, 
malignancy status, and survival status will be provided. 
5.9. 
Pharmacokinetics Analysis 
5.9.1. 
Pharmacokinetic Sampling Schedule  
Blood samples will be collected from all patients at the following schedule: 
Visit 
Study Day 
Cycle 1 Day 1 
1 
Cycle 1 Day 8 
8 
Cycle 2 Day 1 
22 
Cycle 9 Day 1 
169 
Cycle 9 Day 8 
176 
Cycle 10 Day 1 
190 
Cycle 17 Day 1 
337 
Cycle 17 Day 8 
344 
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.    


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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 patients 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 
pharmacokinetic 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. 
5.9.3. 
Pharmacokinetic Analysis 
All ACE-536 serum concentrations will be listed by patient and scheduled time (visit and study).  
Actual dosing/sampling time, sample time relative to first dosing time, visit, and concentration as 
done for the 04 study will be presented in the listing.  
The ACE-536 serum concentrations will be summarized by 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 linear scales.   
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 patient not discontinued:  
 Failure to perform key procedures  
 Study treatment deviation 
 Prohibited concomitant medication 
 GCP related deviation 
5.12. 
Data Handling 
5.12.1. 
Analysis Visit Window 
As specified in Section 5.1, for patients who rollover to Study 06 without interruption, the 
baseline for Study 04 will be used as the baseline for Study 06; for patients who rollover to Study 
06 with interruption, the last value before the first dose of Study 06 will be considered as the 
baseline.  As Study 04 and Study 06 have different schedules of events (Appendix 3 and 


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Appendix 4), analysis window will be derived for summary of endpoints by time points.  
Analysis window determination will be data driven by each parameter.  Detailed rules will be 
specified in the programming specifications. 
5.12.2. 
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.  
5.12.3. 
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: 
 If the year matches the first dose date, then impute the month and day of the first 
dose date. 
 Otherwise, assign January. 
c. If the day is unknown, then: 
 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. 
 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. 
5.12.4. 
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 


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c. if year or the entire date is missing, set to 31 December 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. 
5.12.5. 
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 
5.12.6. 
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 
Baseline hemoglobin for NTD patients will be the average of two or more 
measurements performed during the screening period for base study A536-04 
(for patients without treatment interruption) or study A536-06 (for patients with 
treatment interruption).  Hemoglobin measurements within 2 weeks following 
RBC transfusion will be excluded.  This definition is revised in this SAP to 
reflect the derivation rule implemented for this study. 
Remove Efficacy 
Evaluable (EE) 
population 
ITT provides more conservative estimate of treatment effect.  Since there is no 
ITT population, the word mITT was change to ITT to be consistent with study 04 
analysis population. 


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6. 
REFERENCES 
National Cancer Institute Common Toxicity Criteria for Adverse Events, version 4.0 
(NCI-CTCAE v4.0) 


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7. 
APPENDICES 
7.1. 
Appendix 1 - FACT-An and SF-36 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) 
Patients 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 (i.e. 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. 
 
 


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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, 
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 pre-coded 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 


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 Compute raw scores of each domain by summing up the items in each domain as specified by 
Table 2.  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 in Table 2, 
using the following formula: 
Transformed Score = ((Actual – Lowest)/(Highest – Lowest)) x 100 
 
 
 


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Table 2: 
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 
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 


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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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7.2. 
Appendix 2 - List of Abbreviations 
 
Abbreviation 
Definition 
6MWT 
Six Minute Walk Test 
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 
FACIT-F 
Functional Assessment of Chronic Illness Therapy - Fatigue 
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 


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Abbreviation 
Definition 
ITT 
Intent-to-Treat 
IB 
Investigator’s brochure 
LDH 
Lactate dehydrogenase 
LIC 
Liver Iron Content 
LTB 
Low Transfusion Burden 
LTFU 
Long-term follow-up 
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 
nRBCs 
Nucleated red blood cells 
NTBI 
Non-transferrin bound iron  
PD 
Pharmacodynamic 
PK 
Pharmacokinetic 
PTFU 
Post-treatment follow-up 
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 
SF-36 
Short Form (36-item) Health Survey 
SRT 
Safety review team 
SUSAR 
Suspected unexpected serious adverse reaction 
T1/2 
Elimination half-life 
TGF-β 
Transforming growth factor beta 


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Abbreviation 
Definition 
TIBC 
Total iron binding capacity 
Tmax 
Time to maximum concentration 
ULN 
Upper limit of normal 
WBC 
White blood cell 


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7.3. 
Appendix 3 - Schedule of Events for Study A536-04 
 


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Screen 
Treatment Period 
Follow up period 
Cycle 1 
Cycle 2 
Cycle 3 
Cycle 4 
Cycle 5 
EOT12 
EOS13 
C1D1
2 
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 
 
 
 
 


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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 Protocol 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. 
18For 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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Statistical Analysis Plan 
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Acceleron Pharma Inc. 
7.4. 
Appendix 4 - Schedule of Events for Study A536-06 
 






A536-06 
 
Statistical Analysis Plan 
Confidential and Proprietary  
39 
Acceleron Pharma Inc. 
1 
Screening procedures: Other than informed consent, procedures listed as part of the 28-day screening period are only applicable to patients with treatment interruption.  ECHO or MUGA 
(only required for patients with treatment interruption) can be performed up to 56 days prior to C1D1.  If performed as part of standard of care, ECHO/MUGA does not need to be repeated.  
Patients with treatment interruption will need to qualify per the additional inclusion criteria listed in Section 9.3.2.  For patients without treatment interruption, C1D1 may coincide with 
EOT visit of the base study A536-04.  Procedures that are required to confirm eligibility for patients without treatment interruption can be performed at the base study A536-04 EOT visit 
and used to confirm eligibility prior to dosing on C1D1 of study A536-06.   
2 
Study procedures must be done prior to administration of study drug.  Note that all windows on visits should be determined relative to the date of the previous dose of ACE-536. For 
patients without treatment interruption, C1D1 procedures shaded grey may be conducted as part of the EOT visit for study A536-04 and may not need to be repeated for study A536-06.  
All patients must be assessed for eligibility prior to dosing on C1D1.  C1D1 MRI for LIC (at selected sites) and EMH masses (unless site does not have feasibility to perform the 
assessment) and MRI of the spleen (if no prior splenectomy) can be performed up to 56 days prior to C1D1.  If performed as part of standard of care, MRI for LIC, MRI for EMH and 
spleen size do not need to be repeated.  All screening and C1D1 procedure results required to confirm eligibility must be obtained and reviewed prior to study drug administration in 
A536-06.  On dosing days: Note that the patient dose must be calculated based on the patient’s weight on the day of dosing.  Starting dose level for C1D1, dose modification rules and 
titration rules must be reviewed and implemented prior to dosing as required per protocol (see Section 10.7, Section 10.8 and Section 10.9).  If a dose delay is required per the dose 
modification rules (Section 10.8), the patient will not be dosed.  The patient will return weekly for assessment of hematology results and AEs until the patient is eligible to receive the next 
dose of ACE-536 and start the next cycle. The patient should resume the study at the planned dosing cycle (e.g. if the patient missed a dose at C4D1, then they would resume dosing at 
C4D1 and not skip to C5D1).    
3 
Medical history: Medical history for patients with treatment interruption will include medical events occurring after EOS visit in study A536-04 and prior to C1D1 for study A536-06.  
Medical history for patients without treatment interruption will be taken from study A536-04. 
4 
Quality of life questionnaires are required at C1D1, C5D1, C9D1, C13D1 C17D1, C25D1, C33D1, C41D1, C49D1, C57D1, C65D1, C73D1, C81D1 and EOT. 
5 
Physical exam: Physical exam should also include an optional evaluation of gonadal size of male patients on C1D1, C5D1, C13D1, C22D1, C30D1, C38D1, C46D1, C54D1, C62D1, 
C70D1, C78D1 EOT and EOS. 
6 
Vital signs will include weight, heart rate, and SBP and DBP. 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. 
7 
MRI for liver iron content will be performed at selected sites at C1D1, C9D1 C17D1, C25D1, C33D1, C41D1, C49D1, C57D1, C65D1, C73D1, C81D1 and EOT within a +/- 10-
day window for each scan. A MRI for liver iron content is not required if < 3 months since the previous MRI for liver iron content, unless clinically indicated.  
8 
MRI of the chest and abdomen for EMH mass measurement will be performed for at selected sites at C1D1. If the assessment at C1D1 is negative (no masses), additional scans 
are not required unless clinically indicated. If the assessment at C1D1 is positive (masses present), repeat MRI at C9D1, C17D1, C25D1, C33D1, C49D1, C65D1, C81D1 and EOT 
within a +/- 10-day window for each scan. A MRI of the chest and abdomen for EMH mass measurement is not required if < 3 months since the previous MRI of the chest and 
abdomen for EMH mass measurement, unless clinically indicated 
9 
MRI of the spleen: will be performed at selected sites at C1D1 on patients with no prior splenectomy. If there is no indication of an enlarged spleen on the C1D1 scan, additional 
scans are not required unless clinically indicated. If there is an enlarged spleen on the C1D1 scan, repeat imaging at C9D1 C17D1, C25D1 C33D1, C49D1, C65D1, C81D1 and 
EOT within a +/- 10-day window for each scan.  Patients with clinical signs of a change in spleen size or abnormality should have an abdominal ultrasound or MRI performed as 
needed throughout the study. A MRI for the spleen is not required if < 3 months since the previous MRI for the spleen, unless clinically indicated.     
10 
Leg ulcer assessment: Patients with leg ulcers should have regular assessment of the leg ulcer(s) throughout the study.  Photographs of the leg ulcer(s) pre- and post- dose should 
be provided when available to document any changes in the ulcer(s).   
11 
Serum iron studies: May include serum iron, TIBC, transferrin, soluble transferrin receptor, calculated transferrin saturation, ferritin, NTBI.  The sponsor may perform additional 
sample analysis for biomarkers for exploratory research purposes only.  
12 
Serum folate: Required at screening for patients with treatment interruption to confirm eligibility.  Required for all patients at C1D1, C5D1, C13D1, C21D1, C29D1 C37D1, 
C45D1, C53D1, C61D1, C69D1, C77D1, and EOT. 
13 
Hematology: RBC, nucleated red blood cells (nRBC) (local and central lab), white blood cell (WBC) with differential, hemoglobin, hematocrit, reticulocyte count, platelet count, 
mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution width (RDW).  On 
dosing days, hemoglobin values are to be drawn and resulted (up to 24 hours) prior to dosing (see Section 10.8, Patient Dose Modification Rules).  
14 
Peripheral blood smear: Peripheral blood smears will be prepared centrally from blood sample. 


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Statistical Analysis Plan 
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Acceleron Pharma Inc. 
15 
Chemistry: Sodium, potassium, AST, ALT, 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.  Perform at screening, C1D1, C3D1, C5D1, C9D1, C13D1, C17D1, C21D1, C25D1, C29D1, C33D1, C37D1, 
C41D1, C45D1, C49D1, C53D1, C57D1, C61D1, C65D1, C69D1, C73D1, C77D1, C81D1, EOT and EOS. 
16 
Urinalysis by dipstick analysis (local lab): pH, specific gravity, protein, glucose, ketones, blood, leukocyte esterase, and nitrite, with microscopic examination if indicated.  Urine 
chemistry (central lab): urine chemistries include but are not limited to: microalbumin and creatinine. 
17 
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. 
18 
PK collection is only required on C1D1, C1D8, C2D1, C9D1, C9D8, C10D1, C17D1, and C17D8. 
19 
PD biomarkers: May include hepcidin, GDF15, GDF8, GDF11, activin A and others to be determined.  The sponsor may perform additional sample analysis for biomarkers for 
exploratory research purposes only. 
20 
Hemoglobin electrophoresis: Samples will be collected for central hemoglobin electrophoresis on C1D1, C5D1, C13D1, C21D1, C29D1, C37D1, C45D1, C53D1, C61D1, 
C69D1, C77D1, EOT, and EOS. 
21 
Pregnancy test: (urine or serum) is required for female patients of child bearing potential only. 
22 
Menstrual history: is required for female patients of child bearing potential only 
23 
Transfusion frequency/volume: Transfusion history will be collected from the EOS visit in study A536-04 through the C1D1 visit of A536-06 as available; TD patients will have 
a “pre-transfusion hemoglobin threshold” for requiring transfusion during the study which will be determined based on transfusion history.  For TD patients without treatment 
interruption, baseline pre-transfusion hemoglobin threshold will be the mean of all documented pre-transfusion hemoglobin values during the 12 weeks prior to C1D1 of base study 
A536-04 or study A536-06 (for TD patients with treatment interruption).  During treatment, if the pre-transfusion hemoglobin level 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). 
24 
Adverse events: Patients should be monitored for AEs throughout the study. 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. 
25 
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 EOT visit at the 
time of discontinuation.   
26 
Post-Treatment Follow-Up (PTFU): Should be performed 2 months (± 7 days) after the last dose of ACE-536.  
27 
Long-Term Follow-Up (LTFU): Should be performed every 6 months (± 14 days) after the last dose of ACE-536 for 3 years after the last dose of ACE-536 to monitor for 
presence of malignancy and pre-malignancy, as per standard of care.  
28 
End of Study (EOS): Should be performed 3 years after the last dose of ACE-536.     
29 
Cycles 6 through 17: Procedures and visits for Cycles 6 through 9, 10 through 13, and 14 through 17 follow the same procedures and visits as scheduled for Cycles 2 through 5, 
with some exceptions as noted above. 
30 
Cycles 24 through 87: Patients may not exceed 87 cycles or 1825 days after first dose, whichever occurs first. The last dose of ACE-536 may not be administered after Day 1825, 
regardless of the number of cycles completed. Procedures and visits for Cycles 24 through 27, 28 through 31, 32 through 35, 36 through 39, 40 through 43, 44 through 47, 48 
through 51, 52 through 55, 56 through 59, 60 through 63, 64 through 67, 68 through 71, 72 through 75, 76 through 79, 80 through 83 and 84 through 87 follow the same procedures 
and visits as scheduled for Cycles 20 through 23, with some exceptions as noted above. 
31 
DXA for BMD will be performed at selected sites at C1D1, C9D1, C17D1, C25D1, C33D1, C49D1, C65D1, C81D1 and EOT within a +/- 10-day window for each scan. A DXA 
for BMD is not required if < 3 months since the previous DXA for BMD, unless clinically indicated.     
32 
6MWT for NTD patients will be performed at selected sites at C1D1, C5D1, C9D1, C13D1, C17D1, C25D1, C33D1, C41D1, C49D1, C57D1, C65D1, C73D1, C81D1 and EOT.