Clinical trial NCT04101721
Conditions: Retinopathy of Prematurity
Sponsor: Regeneron Pharmaceuticals
Start date: 2019-10-30; Completion date: 2022-08-18
Enrollment: 127 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The primary objective of the study is to assess the efficacy of aflibercept compared to laser in patients diagnosed with retinopathy of prematurity (ROP). The secondary objectives of the study are to assess the need for a second treatment modality, to assess the recurrence of ROP in the study and to assess the safety and tolerability of aflibercept.

Detailed description:

Arms: arm 1: Patients will receive a single intravitreal (IVT) injection per eligible eye at baseline. arm 2: Patients will undergo laser treatment in each eligible eye at baseline.
Interventions: intervention 1: aflibercept intervention 2: laser photocoagulation
Intervention descriptions: intervention 1: Administered IVT intervention 2: Transpupillary conventional laser will be administered according to standard local procedures.

Protocol documents:
Prot_002.pdf: 
Regeneron Pharmaceuticals, Inc. 
Clinical Study Protocol 
RANDOMIZED, CONTROLLED, MULTI-CENTER STUDY TO ASSESS 
THE EFFICACY, SAFETY, AND TOLERABILITY OF INTRAVITREAL 
AFLIBERCEPT COMPARED TO LASER PHOTOCOAGULATION IN 
PATIENTS WITH RETINOPATHY OF PREMATURITY 
Compound: 
Intravitreal Aflibercept Injection 
Clinical Phase: 
3 
Protocol Number: 
VGFTe-ROP-1920 
Protocol Version: 
VGFTe-ROP-1920 Amendment 1 
Amendment 1 Date of Issue: 
See appended electronic signature page 
Original Date of Issue: 
27 Jun 2019 
Medical /Study Director: 
Regeneron Pharmaceuticals, Inc. 
777 Old Saw Mill River Road 
Tarrytown, NY 10591 
VV-RIM-00083535-1.0 Approved - 29 Jul 2019 GMT-5:00
NCT Number:  NCT04101721
IND Number:  012462 
EudraCT Number:  2019-001764-29 


Clinical Study Protocol 
 
VGFTe-ROP-1920 Amendment 1 
 
Regeneron Pharmaceuticals, Inc. 
Page 2 of 66 
 
CONFIDENTIAL 
AMENDMENT HISTORY 
Amendment 1 
The following table outlines the changes made to the protocol and the affected sections: 
Change and Rationale for Change 
Section Changed 
Updated statistical sections to describe the Per Protocol 
Set (PPS) analysis, which is required for noninferiority 
studies.  Removed reference to pooled analyses (with the 
Bayer sister study) as this is no longer planned. 
Clinical Study Protocol Synopsis: Statistical Plan 
Section 11.3.1 Full Analysis Set 
Section 11.3.2 Per Protocol Set (new section) 
Section 11.4.3.1 Primary Efficacy Analysis 
Clarified that collection of demographic and baseline data 
includes relevant maternal history. 
Section 5.1 Demographic and Baseline Characteristics 
Modified section for simplification and clarity. 
Section 8.1 Investigational and Reference Treatments 
Clarified that the hearing test will be performed once 
only, at any time prior to discharge from the neonatal 
intensive care unit (NICU). 
Table 1 Schedule of Events (moved location of 
footnote #4) 
Section 9.2.1 Procedures Performed Only at the 
Screening/Baseline Visit 
Clarified that a second central nervous system imaging 
will be carried out prior to discharge from the NICU. 
Table 1 Schedule of Events (moved location of 
footnote #5) 
Section 9.2.1 Procedures Performed Only at the 
Screening/Baseline Visit 
Removed description of volumes for blood sampling, as 
this information is referenced in other study document(s). 
Section 9.1.1 Footnotes for the Schedule of Events Table 
(#13 and #14) 
Section 9.2.5 Drug Concentration and Measurements 
Updated protocol to conform with recently-updated 
standard Pharmacovigilance text. 
Section 10.1.1 General Guidelines 
Section 10.2.2 Serious Adverse Event 
Section 10.4 Notifying Health Authorities, Institutional 
Review Board /Ethics Committee, and Investigators 
Added brief description of the RAINBOW study (in 
retinopathy of prematurity) for clarity. 
Section 11.1 Statistical Hypothesis 
Administrative Changes 
Section 1 Introduction 
Section 3.3.1 Known and Expected Benefits 
Section 3.3.2 Potential Risks and Mitigation Measures 
Section 5.2 Efficacy Variables 
Section 9.2.3.1 Ophthalmic Examinations 
Section 9.2.4 Treatment of Overdose 
Section 11.6 Additional Statistical Data Handling 
Conventions 
Minor editorial changes 
Throughout 
 
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Page 3 of 66 
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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
ABR 
Auditory brainstem response 
ADA 
Anti-drug antibody 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
AP-ROP 
Aggressive posterior retinopathy of prematurity 
AST 
Aspartate aminotransferase 
BAER 
Brainstem auditory evoked response 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CONSORT 
Consolidated Standards of Reporting Trials 
CPK 
Creatine phosphokinase 
CRF 
Case report form (electronic or paper) 
CRO 
Contract research organization 
DMC 
Data monitoring committee 
DOB 
Date of birth 
DWFI 
Digital wide-field imaging/images (retina or fundus) 
EC 
Ethics Committee 
EDC 
Electronic data capture 
EOS 
End of study 
FA 
Fluorescein angiography 
FAS 
Full analysis set 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HDL 
High-density lipoprotein 
ICF 
Informed consent form 
ICH 
International Council for Harmonisation 
IOP 
Intraocular pressure 
IRB 
Institutional Review Board 
IVRS 
Interactive Voice Response System 
IVT 
Intravitreal 
IWRS 
Interactive Web Response System 
LDH 
Lactate dehydrogenase 
LDL 
Low-density lipoprotein 
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Page 4 of 66 
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NAb 
Neutralizing antibody 
PCSV 
Potentially clinically significant value 
PD 
Pharmacodynamic 
PK 
Pharmacokinetic 
PPS 
Per protocol set 
RBC 
Red blood cell 
RC 
Reading center 
Regeneron 
Regeneron Pharmaceuticals, Inc. 
ROP 
Retinopathy of prematurity 
SAE 
Serious adverse event 
SAF 
Safety analysis set 
SAP 
Statistical analysis plan 
SAS 
Statistical Analysis System 
SC 
Subcutaneous 
SOC 
System organ class 
SUSAR 
Suspected unexpected serious adverse reaction 
TEAE 
Treatment-emergent adverse event 
US 
United States 
VEGF 
Vascular endothelial growth factor 
VEGF-A 
Vascular endothelial growth factor-A 
VEGFR1 
Vascular endothelial growth factor receptor 1 
VEGFR2 
Vascular endothelial growth factor receptor 2 
WBC 
White blood cell 
 
 
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Clinical Study Protocol 
 
VGFTe-ROP-1920 Amendment 1 
 
 
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Page 5 of 66 
CONFIDENTIAL 
TABLE OF CONTENTS 
AMENDMENT HISTORY .............................................................................................................2 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ..................................................3 
CLINICAL STUDY PROTOCOL SYNOPSIS.............................................................................11 
1. 
INTRODUCTION ......................................................................................................16 
2. 
STUDY OBJECTIVES ..............................................................................................17 
2.1. 
Primary Objective .......................................................................................................17 
2.2. 
Secondary Objectives .................................................................................................17 
2.3. 
Exploratory Objectives ...............................................................................................17 
3. 
HYPOTHESIS AND RATIONALE ..........................................................................18 
3.1. 
Hypothesis ..................................................................................................................18 
3.2. 
Rationale .....................................................................................................................18 
3.2.1. 
Rationale for Study Design .........................................................................................18 
3.2.2. 
Rationale for Dose Selection ......................................................................................18 
3.3. 
Risk-Benefit ................................................................................................................18 
3.3.1. 
Known and Expected Benefits ....................................................................................19 
3.3.2. 
Potential Risks and Mitigation Measures ...................................................................20 
3.3.2.1. 
Ocular Risks ................................................................................................................20 
3.3.2.2. 
Hypersensitivity/Immunogenicity ..............................................................................20 
3.3.2.3. 
Acute Systemic Effects ...............................................................................................20 
4. 
ENDPOINTS ..............................................................................................................21 
4.1. 
Primary and Secondary Endpoints..............................................................................21 
4.1.1. 
Primary Endpoint ........................................................................................................21 
4.1.2. 
Secondary Endpoints ..................................................................................................21 
4.1.3. 
Exploratory Endpoints ................................................................................................21 
5. 
STUDY VARIABLES................................................................................................22 
5.1. 
Demographic and Baseline Characteristics ................................................................22 
5.2. 
Efficacy Variables ......................................................................................................22 
5.3. 
Safety Variables ..........................................................................................................23 
5.4. 
Pharmacokinetic Variables .........................................................................................23 
5.5. 
Immunogenicity Variables ..........................................................................................23 
5.6. 
Biomarker Variables ...................................................................................................23 
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6. 
STUDY DESIGN .......................................................................................................24 
6.1. 
Study Description and Duration .................................................................................24 
6.1.1. 
End of Study Definition ..............................................................................................29 
6.2. 
Planned Interim Analysis ............................................................................................29 
6.3. 
Study Committees .......................................................................................................29 
6.3.1. 
Independent Data Monitoring Committee ..................................................................29 
7. 
SELECTION, WITHDRAWAL, AND REPLACEMENT OF PATIENTS ..............29 
7.1. 
Number of Patients Planned .......................................................................................29 
7.2. 
Study Population .........................................................................................................29 
7.2.1. 
Inclusion Criteria ........................................................................................................29 
7.2.2. 
Exclusion Criteria .......................................................................................................30 
7.2.3. 
Rescreening of Screen Failures ..................................................................................30 
7.3. 
Premature Withdrawal from the Study .......................................................................31 
7.4. 
Replacement of Patients .............................................................................................31 
8. 
STUDY TREATMENTS............................................................................................31 
8.1. 
Investigational and Reference Treatments ..................................................................31 
8.2. 
Rescue Treatments ......................................................................................................32 
8.3. 
Dose Modification and Study Treatment Discontinuation Rules ...............................32 
8.3.1. 
Dose Modification ......................................................................................................32 
8.3.2. 
Study Drug Discontinuation .......................................................................................32 
8.4. 
Method of Treatment Assignment ..............................................................................32 
8.5. 
Masking ......................................................................................................................33 
8.6. 
Treatment Logistics and Accountability .....................................................................33 
8.6.1. 
Packaging, Labeling, and Storage ..............................................................................33 
8.6.2. 
Supply and Disposition of Treatments .......................................................................33 
8.6.3. 
Treatment Accountability ...........................................................................................33 
8.6.4. 
Treatment Compliance ................................................................................................34 
8.7. 
Concomitant Medications ...........................................................................................34 
8.7.1. 
Prohibited Medications ...............................................................................................34 
8.7.2. 
Permitted Medications ................................................................................................34 
8.8. 
Post-study Treatments ................................................................................................35 
9. 
STUDY SCHEDULE OF EVENTS AND PROCEDURES ......................................35 
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9.1. 
Schedule of Events .....................................................................................................35 
9.1.1. 
Footnotes for the Schedule of Events Table ...............................................................38 
9.1.2. 
Early Termination Visit ..............................................................................................39 
9.1.3. 
Unscheduled Visits .....................................................................................................39 
9.2. 
Study Procedures ........................................................................................................40 
9.2.1. 
Procedures Performed Only at the Screening/Baseline Visit .....................................40 
9.2.2. 
Efficacy Procedures ....................................................................................................40 
9.2.2.1. 
Other Ocular Assessments ..........................................................................................40 
9.2.3. 
Safety Procedures .......................................................................................................41 
9.2.3.1. 
Ophthalmic Examinations ..........................................................................................41 
9.2.3.2. 
Physical Examination .................................................................................................41 
9.2.3.3. 
Vital Signs ..................................................................................................................41 
9.2.3.4. 
Hearing Test ................................................................................................................41 
9.2.3.5. 
Laboratory Testing ......................................................................................................41 
9.2.3.6. 
Central Nervous System Imaging ...............................................................................43 
9.2.4. 
Treatment of Overdose ...............................................................................................43 
9.2.5. 
Drug Concentration and Measurements .....................................................................44 
9.2.6. 
Immunogenicity Measurements and Samples ............................................................44 
9.2.7. 
Biomarker Procedures ................................................................................................44 
10. 
SAFETY EVALUATION AND REPORTING .........................................................45 
10.1. 
Recording and Reporting Adverse Events ..................................................................45 
10.1.1. 
General Guidelines .....................................................................................................45 
10.1.2. 
Reporting Procedure ...................................................................................................46 
10.1.3. 
Events that Require Expedited Reporting to Sponsor ................................................46 
10.2. 
Definitions ..................................................................................................................46 
10.2.1. 
Adverse Event .............................................................................................................46 
10.2.2. 
Serious Adverse Event ................................................................................................47 
10.2.3. 
Severity .......................................................................................................................47 
10.2.4. 
Causality .....................................................................................................................48 
10.3. 
Safety Monitoring .......................................................................................................49 
10.4. 
Notifying Health Authorities, Institutional Review Board /Ethics Committee, 
and Investigators .........................................................................................................49 
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11. 
STATISTICAL PLAN................................................................................................50 
11.1. 
Statistical Hypothesis ..................................................................................................50 
11.2. 
Justification of Sample Size........................................................................................50 
11.3. 
Analysis Sets ...............................................................................................................51 
11.3.1. 
Full Analysis Set .........................................................................................................51 
11.3.2. 
Per Protocol Set ..........................................................................................................51 
11.3.3. 
Safety Analysis Set .....................................................................................................51 
11.3.4. 
Pharmacokinetic Analysis Set ....................................................................................51 
11.3.5. 
Immunogenicity Analysis Set .....................................................................................51 
11.4. 
Statistical Methods ......................................................................................................51 
11.4.1. 
Patient Disposition ......................................................................................................51 
11.4.2. 
Demography and Baseline Characteristics .................................................................52 
11.4.3. 
Efficacy Analyses .......................................................................................................52 
11.4.3.1. 
Primary Efficacy Analysis ..........................................................................................52 
11.4.3.2. 
Secondary and Exploratory Efficacy Analyses ..........................................................52 
11.4.4. 
Control of Multiplicity ................................................................................................52 
11.4.5. 
Safety Analysis ...........................................................................................................53 
11.4.5.1. 
Adverse Events ...........................................................................................................53 
11.4.5.2. 
Ocular Safety ..............................................................................................................53 
11.4.5.3. 
Other Safety ................................................................................................................54 
11.4.5.4. 
Treatment Exposure ....................................................................................................54 
11.4.5.5. 
Treatment Compliance ................................................................................................54 
11.4.6. 
Pharmacokinetics ........................................................................................................54 
11.4.6.1. 
Analysis of Drug Concentration Data .........................................................................54 
11.4.6.2. 
Pharmacokinetics and Pharmacokinetics/Pharmacodynamics Analyses ....................54 
11.4.7. 
Analysis of Immunogenicity Data ..............................................................................54 
11.4.8. 
Analysis of Biomarker Data .......................................................................................55 
11.5. 
Interim Analysis ..........................................................................................................55 
11.6. 
Additional Statistical Data Handling Conventions .....................................................55 
11.7. 
Statistical Considerations Surrounding the Premature Termination of a Study .........56 
12. 
QUALITY CONTROL AND QUALITY ASSURANCE .........................................56 
12.1. 
Data Management and Electronic Systems ................................................................56 
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12.1.1. 
Data Management .......................................................................................................56 
12.1.2. 
Electronic Systems ......................................................................................................56 
12.2. 
Study Monitoring ........................................................................................................57 
12.2.1. 
Monitoring of Study Sites ...........................................................................................57 
12.2.2. 
Source Document Requirements ................................................................................57 
12.2.3. 
Case Report Form Requirements ................................................................................57 
12.3. 
Audits and Inspections ................................................................................................57 
12.4. 
Study Documentation .................................................................................................58 
12.4.1. 
Certification of Accuracy of Data ...............................................................................58 
12.4.2. 
Retention of Records ..................................................................................................58 
13. 
ETHICAL AND REGULATORY CONSIDERATIONS ..........................................58 
13.1. 
Good Clinical Practice Statement ...............................................................................58 
13.2. 
Informed Consent .......................................................................................................59 
13.3. 
Patients’ Confidentiality and Data Protection ............................................................59 
13.4. 
Institutional Review Board/Ethics Committee ...........................................................60 
13.5. 
Clinical Study Data Transparency ..............................................................................60 
14. 
PROTOCOL AMENDMENTS ..................................................................................60 
15. 
PREMATURE TERMINATION OF THE STUDY OR CLOSEOUT OF A 
SITE ............................................................................................................................60 
15.1. 
Premature Termination of the Study ...........................................................................60 
15.2. 
Closeout of a Site ........................................................................................................61 
16. 
CONFIDENTIALITY ................................................................................................61 
17. 
FINANCING AND INSURANCE .............................................................................61 
18. 
PUBLICATION POLICY ..........................................................................................61 
19. 
REFERENCES ...........................................................................................................62 
20. 
INVESTIGATOR’S AGREEMENT ..........................................................................64 
SIGNATURE OF SPONSOR’S RESPONSIBLE OFFICERS .....................................................66 
 
 
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LIST OF TABLES 
Table 1: 
Schedule of Events .....................................................................................................36 
 
LIST OF FIGURES 
Figure 1: 
Study Flow Diagram ...................................................................................................25 
Figure 2: 
Aflibercept Treatment, Aflibercept Retreatment, and Rescue Treatment ..................27 
Figure 3: 
Laser Treatment, Laser Retreatment, and Rescue Treatment .....................................28 
 
LIST OF APPENDICES 
APPENDIX 1. FLUORESCEIN ANGIOGRAPHY SUB-STUDY ...........................................65 
 
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Clinical Study Protocol 
 
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CLINICAL STUDY PROTOCOL SYNOPSIS 
Title 
Randomized, Controlled, Multi-Center Study to Assess the Efficacy, Safety, 
and Tolerability of Intravitreal Aflibercept Compared to Laser 
Photocoagulation in Patients with Retinopathy of Prematurity 
Site Location(s) 
 
 
Principal Investigator 
Approximately 60 sites intended from North America, South America, Asia, 
and Europe. 
Each site will have a PI. The treating physician would either be a retinal 
physician or pediatric ophthalmologist treating retinopathy of prematurity 
(ROP) 
Objectives 
The primary objective of the study is to assess the efficacy of aflibercept 
compared to laser in patients diagnosed with ROP. 
The secondary objectives of the study are: 
 
To assess the need for a second treatment modality 
 
To assess the recurrence of ROP in the study 
 
To assess the safety and tolerability of aflibercept 
The exploratory objectives of the study are: 
 
To further characterize the anatomical effects of treatment 
with aflibercept in patients with ROP 
 
To further investigate the study intervention (eg, mode-of-
action-related effects and/or safety) relevant to this disease 
process 
 
To assess the treatment burden of aflibercept and laser 
 
To characterize the concentrations of free and bound 
aflibercept in plasma over time  
 
To describe the potential immunogenicity of aflibercept 
Study Design 
This is a phase 3, multicenter, randomized, 2-arm, open-label clinical study 
to assess the efficacy, safety, and tolerability of intravitreal (IVT) aflibercept 
versus laser in patients with ROP.  The study consists of screening/baseline 
(1 or 2 visits), a treatment period (including potential retreatment and rescue 
treatment), and a final visit at week 52 of chronological age. 
The study also includes an optional fluorescein angiography (FA) sub-study, 
which includes optional FA at baseline, and mandatory FA at week 52 of 
chronological age. 
Study Duration 
The duration of the study for each patient is from baseline to 52 weeks of 
chronological age. 
End of Study Definition 
The end of the study as a whole is defined as the date of the last visit of the 
last patient in the study in all centers in all participating countries. 
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Population 
 
 
Sample Size: 
Approximately 112 patients are planned to be enrolled in a 3:1 ratio 
(approximately 84 in the aflibercept group and approximately 28 in the laser 
group). 
 
Target Population: 
The study population will consist of male and female preterm infants with 
treatment-naïve ROP Zone I Stage 1 plus, 2 plus, 3 non-plus or plus, or 
Zone II Stage 2 plus or 3 plus or AP-ROP according to the International 
Classification for ROP. 
Treatment(s) 
 
 
Aflibercept 
 
Dose/Route/Schedule: 
Patients randomized to aflibercept will receive a single IVT injection of 
aflibercept 0.4 mg/0.01 mL per eligible eye at baseline. 
Thereafter, if required, up to 2 additional IVT injections of aflibercept 
0.4 mg/0.01 mL may be administered in each eye in case both the following 
retreatment criteria are met: 
 
Presence of ROP requiring treatment AND 
 
The interval since the last aflibercept IVT injection is ≥28 days 
Rescue treatment with laser may be performed if 1 of the following 
conditions is met: 
 
Worsening of ROP compared to the examination immediately 
preceding the previous aflibercept injection, during the 27 
days following the aflibercept injection 
 
Presence of ROP requiring treatment after the patient already 
received a total of 3 aflibercept injections 
 
Laser 
Patients randomized to laser will undergo treatment in each eligible eye at 
baseline.  Treatment will be applied to the entire avascular peripheral retina.  
Treatment should be kept well away from the fovea.  Laser ablation should 
be as complete as possible.  It is recommended that fundus photographs 
(digital wide-field imaging [DWFI]) should be obtained right after 
administration of laser treatment to look for “skip” areas, and if found, laser 
should be administered to those areas prior to the investigator deeming the 
laser treatment as “complete.” In case multiple sessions are necessary within 
1 week from baseline, they will be counted as a single treatment. 
Supplementary laser treatments are allowed during the study.  Retreatment 
with laser is allowed if both of the following criteria are met: 
 
Presence of ROP requiring treatment 
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 
Fundus examination reveals area for additional laser treatment 
as judged by the investigator (fundus photography 
recommended to help with adjudication) 
Rescue treatment with aflibercept 0.4 mg/0.01 mL is allowed if the fundus 
examination reveals that laser treatment is complete as judged by the 
investigator and if 1 of the following conditions is met: 
 
Worsening of ROP compared to the most recent pre-laser 
examination 
 
Persistence of ROP requiring treatment 
Patients who initiate aflibercept rescue treatment may receive additional 
aflibercept injections according to the aflibercept group treatment regimen. 
Endpoint(s) 
 
 
Primary: 
The primary endpoint is the proportion of patients with absence of active 
ROP and of unfavorable structural outcomes at week 52 of chronological 
age, as determined by the Investigator.  For patients with both eyes enrolled 
in the study, both eyes must meet the endpoint.  
Unfavorable structural outcomes are defined as retinal detachment, macular 
dragging, macular fold, or retrolental opacity. 
 
Secondary: 
The secondary endpoints are: 
 
Proportion of patients requiring intervention with a second 
treatment modality from baseline to week 52 of chronological 
age 
 
Proportion of patients with recurrence of ROP through 
week 52 of chronological age 
 
The proportion of patients with treatment-emergent adverse 
events (TEAEs) and serious adverse events (SAEs) (ocular 
and systemic) from baseline to week 52 of chronological age 
Procedures and Assessments 
The efficacy procedures/assessments include:  Posterior segment 
assessment, indirect ophthalmoscopy, color fundus photographs using wide-
field digital retinal photography, visual function, retinoscopy, and ocular 
motility. 
The safety procedures/assessments include:  ophthalmic examinations, 
intraocular pressure, physical examination, vital signs, clinical laboratories, 
and central nervous system imaging. 
The study also includes pharmacokinetic, anti-drug antibody, and biomarker 
assessments. 
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Statistical Plan 
Statistical Hypothesis: 
This study will examine the following hypothesis for the primary efficacy 
variable regarding the proportion of patients with absence of active ROP and 
unfavorable structural outcomes at week 52 of chronological age.  Statistical 
testing will be conducted to demonstrate the non-inferiority of the 
aflibercept group to the laser group, with a non-inferiority margin of 5% and 
a significance level of 5% (one-sided test). 
H0:  pt = pc - 5% versus H1:  pt ≥ pc - 5% 
where pt is the true proportion of patients with absence of active ROP and 
unfavorable structural outcomes at week 52 of chronological age for the 
aflibercept group, and pc is the true proportion of patients with absence of 
active ROP and unfavorable structural outcomes at week 52 of 
chronological age for the laser group. 
The proposed non-inferiority margin of 5% is smaller than the smallest 
difference between laser and ranibizumab in the RAINBOW study (a 
phase 3 study comparing the effect of IVT ranibizumab to laser in the 
management of ROP), and not greater than the difference between the 2 
ranibizumab doses. 
Justification of Sample Size: 
From the RAINBOW study, the response rate was 66.1% for the laser group 
and 88.1% for the 0.2 mg ranibizumab group in zone II disease. 
Furthermore, based on the clinical evidence for aflibercept investigator-
initiated studies, the response rates range all the way up to 100% (with at 
least 2 studies demonstrating a 100% response rate in terms of favorable 
anatomic structural outcomes/prevention of unfavorable anatomic structural 
outcomes) with intravitreal aflibercept doses ranging from 0.4 mg to 1 mg. 
An estimated 90% response rate for the aflibercept group and 66.1% 
response rate for the laser group would be a reasonable assumption.  It is 
assumed that the proportion of patients without active ROP will be in line 
with this.  A sample size of 84 patients in the aflibercept group and 
28 patients in the laser group (randomized in a 3:1 ratio) will provide 92% 
power for rejecting the null hypothesis at a 1-sided 5% significance level. 
Statistical Methods: 
Unless stated otherwise, all variables will be analyzed descriptively with 
appropriate statistical methods:  continuous variables by sample statistics 
(ie, mean, standard deviation, median, quartiles, minimum, and maximum), 
and categorical variables by frequencies and percentages. 
Efficacy Analyses: 
The primary and secondary efficacy variable analyses will be conducted on 
the FAS.  The primary analysis is a statistical evaluation of non-inferiority 
of aflibercept vs. laser at week 52 of chronological age, with respect to the 
primary efficacy variable. The non-inferiority margin is set at 5%.  
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Superiority testing will follow if non-inferiority is established.  Sensitivity 
analysis of the primary efficacy endpoint will also be conducted based on 
the per protocol set. 
If data are available from the week 40 of chronological age visit, it will be 
carried forward (LOCF) to the EOS visit for analysis.  If the patient 
discontinued at or before this visit, no data will be carried forward and the 
patient will be considered a non-responder.  For patients with both eyes 
enrolled in the study, both eyes must meet the endpoint.  Patients with only 
one study eye enrolled will be responders if the respective eye responds. 
The statistical analysis will be performed using the Cochran-Mantel-
Haenszel method stratified by baseline ROP status. The 1-sided 95% 
Mantel-Haenszel confidence intervals using normal approximation of the 
difference of response rates between the aflibercept group and the laser 
group will be calculated.  Aflibercept will be considered to be non-inferior 
to laser if the confidence interval of the difference lies entirely above -5%. 
Furthermore, eyes will be considered to be non-responders if rescue 
treatment is given.  The primary analysis for this endpoint will be based on 
the investigator assessment of, and sensitivity analyses based on the central 
RC data will be conducted. 
 
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1. 
INTRODUCTION 
Retinopathy of prematurity (ROP) is a proliferative vascular retinopathy caused by an abnormal 
development of the vascularization of the peripheral retina in premature infants (Mutlu, 2013) 
(Pertl, 2015) (Salman, 2015).  It mainly affects newborns with a preterm gestational age 
(≤32 weeks) and very low birth weight (≤1500 g).  Approximately 10% to 11% of all newborn 
infants are born preterm (Blencowe, 2013) (Hellström, 2013).  Among premature infants, the 
incidence of ROP ranges from 20% to 30%, increasing for younger gestational ages.  The 
prevalence of blindness caused by ROP in developed nations ranges between 6% and 18%, while 
in developing nations the estimate is >20% (Hartnett-1, 2014) (Gilbert, 1997).  Epidemiological 
studies have also identified increased risks for ROP due to genetic variants and environmental 
factors, such as oxygen exposure (Hartnett-2, 2014).  The first wave of ROP cases, in the 1940s 
and 1950s, were identified in preterm infants who were exposed to high levels of supplemental 
oxygen in closed incubators, causing abnormal development of the vascular network in the 
premature retina.  Stricter control of supplemental oxygen led to a decreased incidence of this 
condition (Hellström, 2013).  An English study showed that the incidence of ROP later increased 
from 12.8 per 1000 low birth-weight infants in 1990 to 125.5 per 1000 low-birth-weight infants in 
2011 (Painter, 2015).  This second wave of ROP cases may be attributed to improved survival of 
the most extremely premature infants, introducing a new population of vulnerable newborns more 
susceptible to ROP. 
Vascular endothelial growth factor (VEGF) is up-regulated under ischemic conditions.  As ROP 
is characterized by incomplete vascularization of the retina in premature infants, it has also been 
associated with increased levels of VEGF.  Vascular endothelial growth factor-A (VEGF-A) is a 
member of the VEGF family of angiogenic factors that acts through activation of the receptor 
tyrosine kinases vascular endothelial growth factor receptor 1 (VEGFR1) and vascular endothelial 
growth factor receptor 2 (VEGFR2) to increase mitosis, chemotaxis, and vascular permeability in 
endothelial cells.  Upregulation of VEGF-A due to ischemia in the avascular retina may induce 
pathologic neovascularization and subsequently lead to retinal detachment and blindness, as seen 
in late-stage ROP (Stone, 1996) (Young, 1997).  Aflibercept inhibits these effects of VEGF by 
acting as a soluble decoy receptor that binds VEGF-A with higher affinity than the natural 
receptors and thereby inhibits the binding and activation of these cognate VEGF receptors.  
Aflibercept also inhibits the effects of placental growth factor, which binds only to VEGFR1. 
Placental growth factor can synergize with VEGF-A, enhancing these processes of pathologic 
neovascularization, and is also known to promote leukocyte infiltration and vascular inflammation.  
Based on the its mode of action, aflibercept has a high potential to become an effective treatment 
option for the treatment of ROP, and several reports of off-label clinical use have shown positive 
efficacy outcomes without indicating new safety concerns (Sukgen, 2018) (Salman, 2015) 
(Huang, 2018). 
In addition to the evidence currently available for aflibercept, the efficacy and safety of anti-VEGF 
agents for the treatment of ROP have been compared to laser in several case series and 2 large 
randomized controlled trials (BEAT-ROP and RAINBOW).  The totality of the clinical evidence 
available thus far indicates a trend towards superior prevention of unfavorable anatomical 
outcomes for anti-VEGF agents compared to laser for ROP in central retinal locations (Zone I with 
any ROP stages without plus disease, or Zone I stage 3 without plus disease, or Zone II with 
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stages 2 plus or 3 plus disease), and aggressive posterior ROP (AP-ROP), and establishes the proof 
of concept for this indication (Mintz-Hittner, 2011) (Harder, 2013) (Hwang, 2015). 
Aflibercept has been previously studied for several adult indications characterized by ocular 
neovascularization or increased permeability of the retinal vascular network, and is approved in 
multiple countries for treatment of indications such as diabetic retinopathy (DR), neovascular 
age-related macular degeneration, diabetic macular edema, macular edema secondary to retinal 
venous occlusion and choroidal neovascularization secondary to pathological myopia.  
Considering the consistently positive outcomes provided by aflibercept across these adult 
VEGF-mediated indications, it is deemed appropriate to conduct a study for a pediatric VEGF-
mediated indication for the treatment of ROP. 
Additional background information on the study drug and development program can be found in 
the Investigator’s Brochure. 
2. 
STUDY OBJECTIVES 
2.1. 
Primary Objective 
The primary objective of the study is to assess the efficacy of aflibercept compared to laser in 
patients diagnosed with ROP. 
2.2. 
Secondary Objectives 
The secondary objectives of the study are: 
 To assess the need for a second treatment modality 
 To assess the recurrence of ROP in the study 
 To assess the safety and tolerability of aflibercept 
2.3. 
Exploratory Objectives 
The exploratory objectives of the study are: 
 To further characterize the anatomical effects of treatment with aflibercept in patients 
with ROP 
 To further investigate the study intervention (eg, mode-of-action-related effects and/or 
safety) relevant to this disease process 
 To assess the treatment burden of aflibercept and laser 
 To characterize the concentrations of free and bound aflibercept in plasma over time  
To describe the potential immunogenicity of aflibercept 
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3. 
HYPOTHESIS AND RATIONALE 
3.1. 
Hypothesis 
Intravitreal aflibercept dosed at 0.4 mg will achieve comparable results as assessed by the primary 
endpoint (absence of active ROP and of unfavorable structural outcomes) when compared to laser 
therapy. 
3.2. 
Rationale 
3.2.1. 
Rationale for Study Design 
Laser photocoagulation of the peripheral avascular retina is the current standard of care for the 
treatment of ROP.  Considering aflibercept’s mode of action, and the current clinical evidence 
describing positive outcomes in ROP with off-label use of anti-VEGF in central retinal disease or 
AP-ROP, a randomized clinical trial to compare the ability of these treatments to manage active 
ROP and prevent the development of ocular complications is warranted. 
The substantial amount of existing data describing outcomes of laser in ROP is sufficient to 
provide an accurate understanding of the potential benefits and limitations of this treatment 
modality.  The purpose of the current study is primarily to collect the missing data on the outcomes 
of aflibercept in the treatment of ROP, and for this reason, a 3:1 randomization design is planned.  
Additionally, a fully-masked study of intravitreal (IVT) aflibercept compared to laser is technically 
unfeasible. 
The inclusion and exclusion criteria allow the selection of an appropriate patient population and 
increase the likelihood of producing reliable and reproducible results, while guarding against 
exploitation of vulnerable patients.  The proposed criteria are based on existing clinical knowledge 
and feedback from key opinion leaders involved in treatment of preterm infants with ROP. 
3.2.2. 
Rationale for Dose Selection 
Currently available clinical data (Sukgen, 2018) (Salman, 2015) (Sidorenko, 2018) consistently 
show promising efficacy, with no identification of major safety concerns, when using aflibercept 
doses ranging from 0.4 mg to 1 mg per eye (ie, 1/5 to 1/2 of the 2 mg dose approved for indications 
in adult patients).  In order to limit drug exposure, the lowest dose for which positive efficacy was 
reported (0.4 mg/0.01 mL) was selected for this study.  An injection volume of 0.01 mL is 
considered acceptable for IVT administration in infants. 
3.3. 
Risk-Benefit 
Aflibercept is marketed for the treatment of adult patients with several retinal diseases that are 
characterized by ischemia-induced upregulation of VEGF, are related to pathological 
neovascularization and/or vascular leakage, and can result in retinal thickening and edema, which 
are thought to contribute to vision loss.  The efficacy and safety of IVT aflibercept used in adult 
patients with retinal diseases are well established, and its risk-benefit profile is considered 
favorable. 
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Inhibition of VEGF activity by aflibercept can be expected to result in therapeutic benefit in 
premature infants with ROP, since ROP is also characterized by the pathological development of 
the retinal vasculature and has been associated with VEGF upregulation.  Aflibercept has 
demonstrated efficacy in animal models of pathological ocular neovascularization after systemic 
and IVT administration.  No differences in the mechanism of action of aflibercept in the treatment 
of neovascular ocular diseases between children (eg, ROP) and adults are expected.  There is a 
high and unmet medical need for an effective, safe, and approved treatment for preterm infants 
with vision-threatening ROP, a disease that is a leading cause of blindness in both developing and 
developed countries.  Laser (for the peripheral early stage of ROP) is currently considered a 
standard of care, but fails to achieve a normal retinal structure in up to 25% of ROP patients with 
central retinal disease or AP-ROP.  Additionally, laser is associated with potential risks, including 
irreversible loss of visual field and high myopia.  Vitreoretinal surgery is reserved for advanced 
disease stages with retinal detachment (stages 4 and 5).  Increasing clinical experience from 
off-label use of IVT aflibercept for central ROP suggests positive efficacy outcomes, usually after 
a single injection, with no major safety concerns as currently reported.  Thus, aflibercept has the 
potential to offer improved outcomes in this high-risk, vulnerable premature newborn population 
with ROP. 
3.3.1. 
Known and Expected Benefits 
Aflibercept is expected to provide demonstrable anatomical benefits through regression of ROP 
features while allowing the vascularization to resume its usual development towards the retinal 
periphery, thus preventing unfavorable structural outcomes in approximately 90% of treated 
patients.  Visual function is also expected to improve (preservation of peripheral visual field, 
prevention of loss of best corrected visual acuity, and lower proportion of patients developing high 
myopia). 
Moreover, additional advantages of aflibercept over laser treatment are expected.  An aflibercept 
injection is less time-consuming than laser (an IVT injection is often less than 30 minutes, whereas 
laser treatment may take 2 or more hours).  Laser treatment requires general anesthesia/sedation, 
risks that would be largely avoided when using aflibercept.  Aflibercept treatment may be given 
regardless of the patient's ocular anatomy (while laser treatment would be difficult in patients 
without adequate visualization of the retina, such as in patients with hazy corneas, iris 
neovascularization, or small pupils).  Aflibercept treatment offers the potential for a 
single-injection treatment option and can be administered immediately after diagnosis and 
informed consent by the parent(s)/legally authorized representative(s).  Bilateral cases can be 
treated in a single session.  Upon recurrence of vision-threatening ROP, re-treatment with the same 
dose may be given to each treatment-requiring eye, after injection-free intervals of at least 28 days.  
For laser, once avascular retina ablation is complete, re-treatment may not offer additional benefits.  
In addition to all this, laser treatment is more susceptible to operator-dependent differences with 
the possibility of having significant areas of untreated retina, consequently increasing the risk of 
failure.  Achieving adequate laser treatment depends on the experience and ability of an individual 
practitioner. 
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3.3.2. 
Potential Risks and Mitigation Measures 
Based on the cumulative safety experience with aflibercept for the treatment of adult patients with 
retinal diseases, the potential risks for the study patients include those described below. 
3.3.2.1. 
Ocular Risks 
Ocular risks include intraocular inflammation (including endophthalmitis), retinal tear/ 
detachment, transient intraocular pressure increase, and traumatic cataract. 
Patients with relevant ongoing or significant previous use of systemic steroids at 
immunosuppressive levels are excluded, to mitigate any potential increased risk of local or 
systemic infections, and to address any potential risk of worsening clinical conditions that have 
been historically reported in the context of steroid use in preterm infants, such as more frequent or 
greater incidence rates of necrotizing enterocolitis, cerebral palsy, and gastrointestinal ulcer. 
3.3.2.2. 
Hypersensitivity/Immunogenicity 
Hypersensitivity/immunogenicity (inherent to all therapeutic proteins) is addressed by exclusion 
of relevant patients (see Section 7.2.2). 
3.3.2.3. 
Acute Systemic Effects 
A minimum weight (800 g) at the time of study treatment (see Section 7.2.1) is required.  At this 
minimum weight, a patient in the aflibercept group will receive 1 mg/kg when dosed bilaterally.  
This dose is predicted to increase systemic exposure in preterm infants when compared to adults 
receiving IVT treatment with aflibercept, but to remain below limits determined in adult nAMD 
patients after intravenous dosing of aflibercept with a maximal tolerated dose of 1 mg/kg.  Patients 
will be monitored for any evidence of arterial hypertension, proteinuria, worsening of concomitant 
conditions typical for preterm infants (eg, worsening of intraventricular hemorrhage I or II, other 
non-ocular hemorrhages), or arterial thromboembolic events.  Pharmacokinetic (PK) samples for 
the measurement of systemic aflibercept concentrations (both free aflibercept [the 
pharmacologically active form] and aflibercept bound to VEGF [the inactive form]) will be taken.  
The study population is vulnerable, due to their underlying prematurity, to increased risk for 
multi-organ comorbidities (such as bronchopulmonary dysplasia, intraventricular hemorrhage, 
necrotizing enterocolitis, septic conditions), and developmental delays, compared to term infants.  
Given the high unmet medical need, the potential to more effectively treat ROP with a single 
injection (or possibly up to 3 injections), and the risks associated with laser treatments, the 
potential lifelong benefits to children treated with aflibercept outweigh the potential risks, and the 
expected risk-benefit profile is favorable. 
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4. 
ENDPOINTS 
4.1. 
Primary and Secondary Endpoints 
4.1.1. 
Primary Endpoint 
The primary endpoint is the proportion of patients with absence of active ROP and of unfavorable 
structural outcomes at week 52 of chronological age, as determined by the Investigator.  For 
patients with both eyes enrolled in the study, both eyes must meet the endpoint.  
Unfavorable structural outcomes are defined as retinal detachment, macular dragging, macular 
fold, or retrolental opacity. 
4.1.2. 
Secondary Endpoints 
The secondary endpoints are: 
 Proportion of patients requiring intervention with a second treatment modality from 
baseline to week 52 of chronological age 
 Proportion of patients with recurrence of ROP through week 52 of chronological age 
 The proportion of patients with treatment-emergent adverse events (TEAEs) and 
serious adverse events (SAEs) (ocular and systemic) from baseline to week 52 of 
chronological age 
4.1.3. 
Exploratory Endpoints 
The exploratory endpoints are: 
 The number of aflibercept administrations from baseline to week 52 of chronological 
age 
 The number of laser treatments from baseline to week 52 of chronological age 
 The proportion of patients needing more than 1 aflibercept injection through week 52 
of chronological age 
 The proportion of patients/eyes needing more than 1 laser treatment through week 52 
of chronological age 
 Time to recurrence of ROP 
 Time to intervention with a second treatment modality for ROP or to development of 
unfavorable structural outcomes 
 Completion of vascularization of the peripheral retina to within 1 disc diameter of the 
ora serrata on FP at week 52 of chronological age 
 Assessment of retinal vascularization and leakage on fluorescein angiography (FA) at 
week 52 of chronological age in patients participating in the FA substudy 
 Time to completion of retinal vascularization 
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 Regression of plus disease, regression of pre-retinal vascularized ridge, and progression 
of retinal vascularization beyond the ridge from baseline to week 52 of chronological 
age 
 Progression to stage 4 or 5 ROP from baseline to week 52 of chronological age 
 Evaluation of visual function at week 52 of chronological age 
 Time required to perform aflibercept injection or initial laser treatment 
 Requirement for sedation and/or general anesthesia to complete laser or aflibercept 
injection 
 Number of visits required up to week 52 of chronological age 
 Systemic exposure to free and bound aflibercept (at expected maximum plasma 
concentration and during the elimination period from plasma) determined by sparse 
sampling 
 To assess immunogenicity as incidence of treatment emergent antibodies to aflibercept 
in patients over time. 
5. 
STUDY VARIABLES 
5.1. 
Demographic and Baseline Characteristics 
Baseline characteristics will include standard demography (eg, age, race, weight, height, etc.), 
disease characteristics, medical history, and medication history, as well as relevant maternal 
history, for each patient. 
5.2. 
Efficacy Variables 
The efficacy variables relevant to the primary endpoint are ROP status and ocular anatomy. 
The efficacy variables relevant to the secondary endpoints are: 
 Intervention with a second treatment modality  
 Recurrence of ROP 
The efficacy variables relevant to the exploratory endpoints are: 
 Exposure: 
 Number of aflibercept injections  
 Number of laser treatments 
 Number of rescue treatments 
 Number of visits 
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 Time 
 To recurrence of ROP 
 To intervention with second modality or development of unfavorable structural 
outcomes 
 Needed to perform aflibercept injection and initial laser treatment 
 Assessment of retinal vasculature and ROP status with fundus exam, FP and FA (for 
those participating in FA substudy), including time to completion of retinal vasculature 
 Assessment of visual function 
 Need for sedation and/or general anesthesia 
5.3. 
Safety Variables 
The safety variables are: 
 Adverse events (TEAEs and SAEs) 
 Ophthalmic examinations 
 Physical examinations 
 Vital signs 
 Clinical laboratories 
 Central nervous system imaging 
5.4. 
Pharmacokinetic Variables 
The PK variable is the concentration of free or bound aflibercept.in plasma at each time point.  
Samples in this study will be collected using a sparse sampling schedule (eg, only 1 blood sample 
for drug concentration assessment will be collected at any single clinic visit).  These sampling time 
points are specified in Table 1. 
5.5. 
Immunogenicity Variables 
Anti-drug antibody variables will include ADA status (positive or negative), titer, and neutralizing 
antibody (NAb) status for analyzed samples collected at the time points specified in Table 1. 
5.6. 
Biomarker Variables 
The biomarker variables may include those relevant to diagnostic, safety, pharmacodynamics, 
monitoring, or potentially predictive biomarkers. 
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6. 
STUDY DESIGN 
6.1. 
Study Description and Duration 
This is a phase 3, multicenter, randomized, 2-arm, open-label clinical study to assess the efficacy, 
safety, and tolerability of IVT aflibercept versus laser in patients with ROP.  The study consists of 
screening/baseline (1 or 2 visits), a treatment period (including potential retreatment and rescue 
treatment), and a final visit at week 52 of chronological age (Figure 1). 
The study also includes an optional fluorescein angiography (FA) sub-study (Appendix 1), which 
includes optional FA at baseline, and mandatory FA at week 52 of chronological age. 
 
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Figure 1: 
Study Flow Diagram 
 
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Successful screening requires the presence of treatment-naïve ROP classified by the investigator 
according to the International Classification for Retinopathy of Prematurity in at least 1 eye with 
1 of the following retinal findings: 
 Zone I Stage 1 plus, 2 plus, 3 non-plus or 3 plus, or 
 Zone II Stage 2 plus or 3 plus, or 
 AP-ROP 
One or both eyes can be treated according to the investigator’s assessment of the study’s eligibility 
criteria.  If both eyes are eligible, they will be assigned to the same treatment group.  The second 
eye of patients who start the study with only 1 eligible eye should be kept under observation 
according to the local ROP screening guidelines or at every study visit, whichever is more frequent.  
Second eyes that develop ROP requiring treatment during the study should receive treatment 
according to the randomization assignment of the first eye.  If a second eye is treated within 
8 weeks of the initial treatment of the first eye, the second eye will be included in the efficacy 
analysis. 
Color fundus photography with digital wide-field images (DWFI) of the retina will be taken before 
any treatment is applied.  These images will be submitted to a central reading center (RC) as soon 
as possible for confirmation of ROP staging, and if deemed eligible, treatment should be 
administered.  However, due to the urgency of treatment for this medical condition, in situations 
where the investigator considers that awaiting a response from the RC and thus delaying treatment 
may be detrimental to the outcome, treatment is allowed to be administered immediately after the 
images are acquired, before availability of the RC confirmation of ROP staging.  In this case, the 
investigator's assessment of the images will be used in stratification. 
Patients will be randomized 3:1 to treatment with either aflibercept injection or laser, respectively. 
Aflibercept group: 
Patients randomized to aflibercept will receive a single IVT injection of aflibercept 
0.4 mg/0.01 mL per eligible eye at baseline. 
Thereafter, if required, up to 2 additional IVT injections of aflibercept 0.4 mg/0.01 mL may be 
administered in each eye in case both the following retreatment criteria are met (Figure 2): 
 Presence of ROP requiring treatment AND 
 The interval since the last aflibercept IVT injection is ≥28 days 
Rescue treatment with laser may be performed if 1 of the following conditions is met (Figure 2): 
 Worsening of ROP compared to the examination immediately preceding the previous 
aflibercept injection, during the 27 days following the aflibercept injection 
 Presence of ROP requiring treatment after the patient already received a total of 
3 aflibercept injections 
Once rescue treatment is applied, treatment with the patient’s randomized treatment cannot be 
reinitiated. 
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Figure 2: 
Aflibercept Treatment, Aflibercept Retreatment, and Rescue Treatment 
 
Once rescue treatment is applied, treatment with the patient’s randomized treatment cannot be reinitiated. 
 
Laser Group: 
Patients randomized to laser will undergo treatment in each eligible eye at baseline.  Treatment 
will be applied to the entire avascular peripheral retina.  Treatment should be kept well away from 
the fovea. Laser ablation should be as complete as possible.  It is recommended that fundus 
photographs (DWFI) should be obtained right after administration of laser treatment to look for 
“skip” areas, and if found, laser should be administered to those areas prior to the investigator 
deeming the laser treatment as “complete.”  In case multiple sessions are necessary within 1 week 
from baseline, they will be counted as a single treatment. 
Supplementary laser treatments are allowed during the study.  Retreatment with laser is allowed if 
both of the following criteria are met (Figure 3): 
 Presence of ROP requiring treatment 
 Fundus examination reveals area for additional laser treatment as judged by the 
investigator (fundus photography recommended to help with adjudication) 
Rescue treatment with aflibercept 0.4 mg/0.01 mL is allowed if the fundus examination reveals 
that laser treatment is complete as judged by the investigator and if 1 of the following conditions 
is met (Figure 3): 
 Worsening of ROP compared to the most recent pre-laser examination 
 Persistence of ROP requiring treatment 
Patients who initiate aflibercept rescue treatment may receive additional aflibercept injections 
according to the aflibercept group treatment regimen. 
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Figure 3: 
Laser Treatment, Laser Retreatment, and Rescue Treatment 
 
Once rescue treatment is applied, treatment with the patient’s randomized treatment cannot be reinitiated. 
 
Both Groups: 
Once rescue treatment is applied to an eye, treatment in that eye with the patient’s randomized 
treatment cannot be reinitiated.  However, the fellow eye can still receive the patient’s randomized 
study intervention, if retreatment criteria are met. 
Patients will have mandatory evaluations at regular intervals during the study at the time points 
specified in Table 1.  Patients receiving retreatment and/or rescue treatment will have mandatory 
evaluations 1 day, 1 week, and 4 weeks after the retreatment/rescue treatment to a given eye. 
Acquisition and submission of DWFIs (eg, RetCam) to the RC is required at baseline and at 
weeks 1, 2, 3, 4, 12, and 24, at week 52 of chronological age, before each retreatment and/or rescue 
treatment, and 1 week and 4 weeks after each retreatment/rescue treatment.  Safety will be assessed 
through the evaluation of adverse events (AEs), ophthalmic and physical examinations (including 
assessment of acute tolerability post-dosing), vital signs, and laboratory tests.  Pharmacokinetic 
evaluations will investigate plasma concentrations of free and bound aflibercept.  The potential 
emergence of ADAs will also be evaluated by serum sampling at baseline and week 12. 
Additional optional evaluation visits may be performed in accordance with the usual standard of 
care, as medically needed. 
A phase 3b extension study is planned to assess the long-term outcomes of patients who received 
study intervention in this study.  All treated patients will be offered an opportunity to participate 
until they are 5 years of age to assess ocular effects, and clinical and neurodevelopmental 
outcomes. 
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6.1.1. 
End of Study Definition 
A patient is considered to have completed the study if he/she has completed all phases of the study 
including the last visit at week 52 of chronological age. 
The end of the study as a whole is defined as the date of the last visit of the last patient in the study 
in all centers in all participating countries. 
Primary completion is defined as the date of the last visit of the last patient for the primary 
outcome. 
6.2. 
Planned Interim Analysis 
No interim analysis is planned. 
6.3. 
Study Committees 
6.3.1. 
Independent Data Monitoring Committee 
The aim of the safety assessments is to determine if the study shows unacceptable risks for the 
patients, and to discontinue the study if necessary.  The assessments will be performed by an 
independent data monitoring committee (DMC).  Detailed information regarding the DMC 
procedures will be explained in a separate DMC charter. 
7. 
SELECTION, WITHDRAWAL, AND REPLACEMENT OF 
PATIENTS 
7.1. 
Number of Patients Planned 
Approximately 112 patients are planned to be enrolled in a 3:1 ratio (approximately 84 in the 
aflibercept group and approximately 28 in the laser group). 
7.2. 
Study Population 
The study population will consist of male and female preterm infants with treatment-naïve ROP 
Zone I Stage 1 plus, 2 plus, 3 non-plus or plus, or Zone II Stage 2 plus or 3 plus or AP-ROP 
according to the International Classification for ROP. 
7.2.1. 
Inclusion Criteria 
A patient must meet the following criteria at screening and baseline to be eligible for inclusion in 
the study: 
1. Gestational age at birth ≤32 weeks or birth weight ≤1500 g 
2. Patients with treatment-naïve ROP classified according to the International Classification 
for ROP in at least one eye as: 
 Zone I Stage 1 plus, or 2 plus, or 3 non-plus or 3 plus, or 
 Zone II Stage 2 plus or 3 plus, or 
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 AP-ROP 
3. Weight at baseline (day of treatment) ≥800 g 
4. Male or female 
5. Signed informed consent from parent(s)/legally authorized representative(s) as described 
in Section 13.2, which includes compliance with the requirements and restrictions listed in 
the informed consent form (ICF) and in this protocol. 
7.2.2. 
Exclusion Criteria 
A patient who meets any of the following criteria will be excluded from the study: 
1. Known or suspected chromosomal abnormality, genetic disorder, or syndrome 
2. Previous exposure to any IVT or systemic anti-VEGF agent, including maternal exposure 
during pregnancy and/or during breastfeeding 
3. Clinically significant neurological disease (eg, intraventricular hemorrhage grade 3 or 
higher, periventricular leukomalacia, congenital brain lesions significantly impairing optic 
nerve function, severe hydrocephalus with significantly increased intracranial pressure) 
4. Pediatric conditions rendering the infant ineligible for study intervention at baseline or for 
repeated blood draws as evaluated by a neonatal intensive care unit specialist and a study 
ophthalmologist 
5. Presence of active ocular infection within 5 days of the first treatment 
6. Advanced stages of ROP with partial or complete retinal detachment (ROP stage 4 and 
stage 5) 
7. ROP involving only Zone III 
8. Ocular abnormalities that may interfere with the administration of study intervention or 
assessment of the study primary endpoint 
9. Postnatal treatment with oral or intravenous corticosteroids at an equivalent dose of 
prednisone ≥1 mg/kg/day for >2 weeks within 14 days of the first study intervention 
10. Previous surgical or nonsurgical treatment for ROP (IVT anti-VEGF injection, ablative 
laser therapy, cryotherapy, and vitrectomy) 
11. Participation of the patient or the mother in other clinical trials requiring administration of 
investigational treatments (other than vitamins and minerals) at the time of screening, or 
within 30 days or 5 half-lives of administration of the previous study drug, whichever is 
longer 
7.2.3. 
Rescreening of Screen Failures 
Screen failures are patients for whom informed consent has been obtained but who do not 
subsequently enter the study.  A minimal set of screen failure information is required to ensure 
transparent reporting of screen failure patients to meet the Consolidated Standards of Reporting 
Trials (CONSORT) publishing requirements and to respond to queries from regulatory authorities.  
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Minimal information includes demography, screen failure details, eligibility criteria, and any 
serious pretreatment events. 
In general, re-starting the defined set of screening procedures to enable the “screen failure” 
patient’s participation at a later time point is not allowed.  Thus, in general, participation of an 
initial “screen failure” patient at a later time point is not acceptable, except if the screening failure 
was triggered by missing inclusion criteria 2 and/or 3, or by meeting exclusion criteria 4, 5, 8, 9, 
and/or 11, or if the inclusion/exclusion criteria preventing the patient’s initial attempt to participate 
have been changed via protocol amendment. 
Under any of the above exceptions, a patient may only be re-screened once, and only within 
10 days of the start of the first screening period. 
To be eligible, rescreened patients must meet all selection criteria at the re-screening visit.  In any 
case, the investigator has to ensure that the repeated screening procedures do not expose the patient 
to an unjustifiable health risk.  Also, for rescreening, the patient’s parent(s)/legally authorized 
representative(s) have to sign a new ICF, even if it was not changed after the patient’s previous 
screening. 
Rescreened patients should be assigned a new patient number. 
7.3. 
Premature Withdrawal from the Study 
A patient's parent(s)/legal guardian(s) has the right to withdraw the patient from the study at any 
time, for any reason, and without repercussion. 
The investigator and/or sponsor have the right to withdraw a patient from the study if it is no longer 
in the interest of the patient to continue in the study, or if the patient’s continuation in the study 
places the scientific outcome of the study at risk (eg, if a patient is not able to follow study 
procedures).  An excessive rate of withdrawals would render the study uninterpretable; therefore, 
unnecessary withdrawal of patients should be avoided. 
Parent(s)/legal guardian(s) of patients who are withdrawn prematurely from the study will be asked 
for the patient to complete the early termination visit, as described in Section 9.1.2. 
7.4. 
Replacement of Patients 
Patients prematurely discontinued from the study will not be replaced. 
8. 
STUDY TREATMENTS 
8.1. 
Investigational and Reference Treatments 
Patients will be randomized 3:1 to receive treatment with an IVT injection of aflibercept 0.4 mg 
or laser. 
Aflibercept (at a concentration of 40 mg/mL) will be supplied in sterile, sealed, 3 ml glass vials.  
The injection volume will be 10 µL (0.01 mL) and will be administered to the patients by IVT 
injection.  Instructions on dose preparation are provided in the pharmacy manual. 
See Section 6.1 for aflibercept dosing strategies. 
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Transpupillary conventional laser will be given following topical anesthesia, sedation, or general 
anesthesia, with appropriate respiratory support as required (eg, laryngeal mask, endotracheal 
intubation, or similar), and administered according to standard local procedures.  The pupil of the 
eye to be treated must be dilated (mydriasis) with 2 or 3 drops of mydriatic agent(s) applied 
topically to the eye, according to local practice. 
Laser power settings should be defined according to the investigator’s practice and applicable 
medical standards at the site.  The treatment should cover the entire avascular retina, using a 
confluent laser pattern and extending to the ora serrata. 
See Section 6.1 for laser treatment strategies. 
8.2. 
Rescue Treatments 
For patients randomized to aflibercept, rescue treatment with laser may be performed.  For patients 
randomized to laser, rescue treatment with aflibercept may be performed.  The criteria for the 
allowance of rescue treatment are specified in Section 6.1.  Use of rescue treatment in either eye 
must be recorded on the respective electronic case report form (eCRF) page. 
All patients requiring rescue treatment will be counted as non-responders in the primary endpoint.  
Nevertheless, the patients should be followed to assess the efficacy and safety outcomes after 
rescue treatment. 
8.3. 
Dose Modification and Study Treatment Discontinuation Rules 
8.3.1. 
Dose Modification 
Dose modification for an individual patient is not allowed. 
8.3.2. 
Study Drug Discontinuation 
Patients who permanently discontinue from study drug should be encouraged to remain in the 
study.  Those who agree and do not withdraw from the study will be asked to return to the clinic 
for all remaining study visits per the visit schedule. 
Patients who permanently discontinue from study drug and who opt to withdraw from the study 
will be asked to complete study assessments, per Section 9.1.2. 
8.4. 
Method of Treatment Assignment 
Eligible patients will be randomized 3:1 to receive either treatment with aflibercept or laser, 
respectively, stratified by ROP classification in Zone I, Zone II, or AP-ROP according to 
investigator assessment.  If both eyes meet the inclusion criteria of the study after screening, the 
eye with the more severe disease will be considered for stratification. 
Treatment allocation will be done according to a computer-generated randomization list specified 
by the sponsor's responsible statistician and provided by the sponsor's randomization management 
group.  To prevent imbalances, the randomization result of patients who do not complete baseline 
treatment will be assigned to a subsequent patient once it is known that the baseline treatment was 
not completed. 
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Patients will be centrally assigned to randomized study intervention using an Interactive Voice 
Response System/Interactive Web Response System (IVRS/IWRS).  Before the study is initiated, 
the telephone number and call-in directions for the IVRS and/or the log in information and 
directions for the IWRS will be provided to each site.  The investigator will provide the 
IVRS/IWRS with study center identification, the patient´s date of birth (DOB), gender, and weight 
at birth.  The complete DOB (day, month, and year) will be entered if allowed by local regulation 
because it is needed in order to correctly determine the participant’s chronological and corrected 
age during the study.  Because of the particular population in this study, absence of an exact DOB 
will cause a 30-day deviation, resulting in possible misinterpretation of results and confounding 
the ability to understand the outcomes, as this is usually compared with controls matched for the 
same corrected gestational age.  Additional details are documented in the IVRS/IWRS instruction 
manuals.  Study intervention will be dispensed at the study visits summarized in Table 1. 
8.5. 
Masking 
This is an open-label study where the study sites, the study team and the patient and patient’s 
parent(s)/legally authorized representative(s) know the treatment the patient is being given.  
Potential bias will be reduced by central randomization and objective endpoints.  Any aggregated 
treatment-specific data will be kept strictly confidential and will not be communicated to 
investigators. 
8.6. 
Treatment Logistics and Accountability 
8.6.1. 
Packaging, Labeling, and Storage 
Open-label study drug will display the product lot number on the label.  Each vial of study drug 
will be labeled per country requirements. 
Study drug will be stored at the site at a temperature of 2ºC to 8ºC; storage instructions will be 
provided in the pharmacy manual. 
8.6.2. 
Supply and Disposition of Treatments 
Study drug will be shipped at a temperature of 2ºC to 8ºC to the investigator or designee at regular 
intervals or as needed during the study.  At specified time points during the study (eg, interim site 
monitoring visits), at the site close-out visit, and following drug reconciliation and documentation 
by the site monitor, all opened and unopened study drug will be destroyed/ returned to the sponsor 
or designee. 
8.6.3. 
Treatment Accountability 
All drug accountability records must be kept current. 
The investigator must be able to account for all opened and unopened study drug.  These records 
should contain the dates, quantity, and study medication 
 dispensed to each patient 
 returned from each patient (if applicable), and 
 disposed of at the site or returned to the sponsor or designee. 
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All accountability records must be made available for inspection by the sponsor and regulatory 
agency inspectors; photocopies must be provided to the sponsor at the conclusion of the study. 
8.6.4. 
Treatment Compliance 
Study intervention will be administered by a qualified ophthalmologist.  Details of aflibercept 
injection and of the laser procedure will be recorded in the eCRF (eg, time required, type of 
anesthesia, presence or absence of endotracheal intubation, treatment site). 
All drug compliance records must be kept current and made available for inspection by the sponsor 
and regulatory agency inspectors. 
8.7. 
Concomitant Medications  
All medications the patient received prior to visit 1 and any medication or vaccine (including any 
sedation, anesthesia, eye drops used for the study procedures, blood-derived products, prescription 
or over-the-counter medicines, probiotics, vitamins, and herbal supplements) that the patient is 
receiving at the time of enrollment or receives during the study should be recorded along with: 
 The reason for use 
 Dates of administration including start and end dates 
 Dosage information including dose and frequency 
The use of medications, recreational drugs, or similar substances during pregnancy will be 
recorded on the Maternal Prior Medication section on the eCRF at screening (eg, alcohol, tobacco, 
antibiotics, corticosteroids, vaccination, supplements).  For breastfeeding mothers, all maternal 
medication or similar products taken after the umbilical cord clamping will be recorded in the 
Maternal Concomitant Medication section of the eCRF only if the transfer of these products into 
human milk is feasible according to the investigator’s judgment.  The Medical Monitor should be 
contacted if there are any questions regarding concomitant or prior therapy. 
8.7.1. 
Prohibited Medications 
Exposure to any IVT or systemic anti-VEGF agent is not allowed in patients or in mothers who 
are breastfeeding. 
8.7.2. 
Permitted Medications 
The use of phototherapy lamp for treating jaundice is allowed.  Appropriate eye protection must 
be provided to the patient.  The use of phototherapy for jaundice will be documented in the eCRF, 
including the use of eye protection. 
The use of oxygen supplementation will be recorded in the corresponding eCRF section with 
special consideration of the method, concentration, duration of administration and the patient´s 
oxygen saturation by pulse oximetry once the supplementation is stable.  In the last visit of the 
study, it will be recorded if oxygen supplementation is required.  Standard of care treatment for 
other conditions affecting patients is permitted. 
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8.8. 
Post-study Treatments 
A phase 3b extension study is planned to assess the long-term outcomes of patients who received 
study intervention in this study and accept participation in the extension study.  No study treatment 
will be required or provided by the sponsor in the extension study.  In case any treatment for ROP 
is required during the extension study, the site’s standard of care should be administered.  Patients 
will undergo safety and efficacy assessments until 5 years of age. 
The baseline visit of the extension study can be conducted concomitantly with the week 52 
chronological age visit of this study. 
9. 
STUDY SCHEDULE OF EVENTS AND PROCEDURES 
9.1. 
Schedule of Events 
Study assessments and procedures are presented by study period and visit in Table 1. 
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Table 1: 
Schedule of Events 
Visit Number1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
EOS 
Retreatment or 
rescue treatment follow-up visits 
Visit Name1 
SCR 
BL 
D1 
W1 
W2 
W3 
W4 
W6 
W8 W10 W12 W16 W20 
W24 
W40 
CA 
W52 
CA 
TRT 
Visit 
D1 
FUP 
1W 
FUP 
4W 
FUP 
Visit Window 
-- 
-- 
+2D ±3D ±3D ±3D +7D ±7D ±7D ±7D ±7D ±7D ±7D 
±7D 
±10D 
±10D 
-- 
+2D 
±3D 
+7D 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In/exclusion criteria 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enroll (IVRS/IWRS) 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographic 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history2 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prior/concomitant 
medication3 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AEs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Body weight 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Head circumference and 
body length 
X 
 
 
 
 
 
 
 
X 
 
 
X 
 
X 
X 
X 
 
 
 
 
Hearing test 
X4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CNS imaging 
X5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical exam6 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs7 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Anterior segment 
examination8 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Binocular indirect 
ophthalmoscopy9 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Tonometry10 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Digital wide-field retinal 
imaging11 
 
X 
 
X 
X 
X 
X 
 
 
 
X 
 
 
X 
 
X 
X 
 
X 
X 
Fluorescein angiography 
(for optional sub-study)12 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Blood sample for ADA 
(aflibercept group only)13 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
Blood sample for PK 
(aflibercept group only)14 
 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Visit Number1 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
EOS 
Retreatment or 
rescue treatment follow-up visits 
Visit Name1 
SCR 
BL 
D1 
W1 
W2 
W3 
W4 
W6 
W8 W10 W12 W16 W20 
W24 
W40 
CA 
W52 
CA 
TRT 
Visit 
D1 
FUP 
1W 
FUP 
4W 
FUP 
Visit Window 
-- 
-- 
+2D ±3D ±3D ±3D +7D ±7D ±7D ±7D ±7D ±7D ±7D 
±7D 
±10D 
±10D 
-- 
+2D 
±3D 
+7D 
 
Hematology, clinical 
chemistry, urinalysis15 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine protein test16 
 
X 
X 
 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Aflibercept injection (for 
patients randomized to 
active)17 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Laser treatment (for 
patients randomized to 
control)18 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Visual function 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
 
 
 
Refraction 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Ocular extrinsic motility 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
 
 
 
Abbreviations  CA = chronological age 
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9.1.1. 
Footnotes for the Schedule of Events Table 
1. Additional visits may be performed depending on the investigator’s assessment of the 
patient’s response to treatment, as part of local standard of care.  Visits 1 and 2 can be on 
same day or within 10 days of each other.  If treatment cannot take place at visit 2, it can 
be administered within 3 days of visit 2.  For Visits 3 to 14, the intervals are based on the 
date of initial treatment.  The intervals of the rescue/retreatment follow-up visits are based 
on the date of rescue/retreatment.  A retreatment/rescue follow-up visit may be combined 
with a regularly scheduled visit.  If a patient’s second eye qualifies for treatment during the 
study, both eyes can follow the visit schedule of the first eye after the retreatment/rescue 
treatment follow-up visit schedule is completed for the second eye. 
2. Maternal and patient medical history will be recorded. 
3. Includes recording of oxygen supplementation.  At screening, includes any of the 
medications listed in Section 8.7 given to mother during pregnancy and breastfeeding. 
4. Hearing test to be performed once, at any time prior to discharge from neonatal intensive 
care unit (NICU). 
5. Imaging is not required at screening if results from an imaging exam performed within the 
previous 10 days are available and there are no new neurological sign/symptoms.  A second 
CNS imaging is to be carried out prior to discharge from the NICU. 
6. Includes cardiovascular, respiratory, gastrointestinal, and neurological systems according 
to local general practice and aiming to evaluate overall health. 
7. Temperature, blood pressure (before study treatment, if applicable), respiratory, and pulse 
rate. 
8. Can be done using indirect ophthalmoscopy or portable slit lamp. 
9. The pupils must be sufficiently dilated to allow examination of all ROP features.  
Unfavorable structural outcome is defined as retinal detachment, macular dragging, 
macular fold, or retrolental opacity. 
10. Only in patients receiving aflibercept.  The IOP will be measured in both eyes prior to the 
injection, and at least once post-injection (only in treated eyes). 
11. Baseline imaging will be done prior to study treatment on the treatment day or up to 2 days 
prior to treatment.  Imaging at week 24 and/or week 52 of chronological age may not be 
required if the following conditions are met:  the reading center assessment of 
morphological outcomes on the week 12 image is in agreement with indirect 
ophthalmoscopy performed by the investigator, and the patient shows poor tolerance for 
the imaging procedure, preventing acquisition of images with adequate quality.  Cases of 
rescue/retreatment require additional imaging before any treatment (on the treatment day 
or up to 2 days prior to treatment) as well as 1 week and 4 weeks following rescue/ 
retreatment. 
12. Optional at baseline; mandatory at week 52 of chronological age (visit 16). 
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13. Only applicable for patients treated with aflibercept at baseline.  One blood sample will be 
taken at baseline prior to dosing.  At week 12, another blood sample will be taken prior to 
dosing to detect anti-drug antibodies (ADAs) and, if applicable, the occurrence of potential 
neutralizing antibodies (NAb) (if collection is not possible, the week 12 sample can be 
taken at week 16). 
14. Only applicable for patients treated with aflibercept at baseline.  One blood sample each 
for plasma concentrations of study drug will be taken on day 1 (approximately 24 hours 
after dosing), day 14, and day 28.  Blood pressure must be measured before the PK sample 
is taken.  If a patient’s second eye is deemed eligible for treatment and is treated with 
aflibercept at visits 3, 5, or 7, the PK sample is taken before treatment. 
15. A sample is not needed if results from laboratory tests within 8 days prior to screening are 
available and there was no change in the clinical situation from the time of the sample to 
the screening visit. 
16. The baseline sample can be taken up to 2 days prior to baseline.  The day 1 sample will be 
approximately 24 hours after study treatment. 
17. The injection should be performed in both eyes on the same day, if applicable.  After initial 
treatment, each study eye may receive up to 2 additional treatments (with a minimum 
interval of 28 days between injections) only if retreatment criteria are met.  After 
retreatment, the retreatment/rescue treatment follow-up visits are also required.  Blood 
pressure must be measured before the injection.  Details of each injection will be recorded 
in the eCRF. 
18. Multiple sessions performed within 1 week after baseline to complete the procedure will 
be counted as a single treatment.  Each study eye may receive retreatment only if the 
retreatment criteria are met.  After retreatment, the retreatment/rescue treatment follow-up 
visits are also required.  Blood pressure must be measured before laser. 
9.1.2. 
Early Termination Visit 
Patients who are withdrawn from the study before the primary endpoint visit (week 52 of 
chronological age) will be asked to return to the clinic:  once for an early termination visit 
consisting of the end of study assessments described in Table 1. 
9.1.3. 
Unscheduled Visits 
All attempts should be made to keep patients on the study schedule.  Unscheduled visits may be 
necessary to repeat testing following abnormal laboratory results, for follow-up of AEs, 
management and/or follow-up of procedures performed in the observation phase of the study, or 
for any other reason, as warranted. 
Unscheduled visits for observation follow the list of assessments used for visit 8 (week 6).  In case 
treatment is required, the assessments listed in the retreatment visit will be performed. 
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9.2. 
Study Procedures 
9.2.1. 
Procedures Performed Only at the Screening/Baseline Visit 
Procedures performed only at the screening/baseline visits are:  Informed consent, inclusion/ 
exclusion criteria, demographics, medical history, and collection of clinical laboratory samples 
(Table 1). 
9.2.2. 
Efficacy Procedures 
All ophthalmic evaluations will be conducted according to the schedule detailed in Table 1. 
Posterior segment assessment to confirm ROP staging or resolution will be evaluated by the 
investigator by binocular indirect ophthalmoscopy or wide-field digital retinal photography (eg, 
RetCam, Phoenix ICON Camera). 
Indirect ophthalmoscopy will be performed according to local medical practice and applicable 
medical standards at the site (eg, usually using a head-mounted light source and a 25- 28, or 
30-diopter lens).  For this examination, the pupil of the eye must be dilated (mydriasis) with topical 
application of mydriatic eye drops to the eye. 
Clinically significant abnormal findings will be reported as AEs in the eCRF. 
Color fundus photographs using wide-field digital retinal photography have to be taken at the time 
points specified in Table 1. 
All images taken by wide-field digital retinal imaging photography during the study will be 
submitted to a central reading center to confirm the ROP staging or resolution, and analysis of 
efficacy data.  All images are part of the source data for the study and must be retained by the 
investigator site.  A detailed protocol for color fundus photographs image acquisition and 
transmission can be found in the respective manual. 
9.2.2.1. 
Other Ocular Assessments 
With all ophthalmological examinations, abnormalities of the retina or optic nerve as well as 
unfavorable ocular structural outcomes in each eye will be assessed.  All of the following ocular 
assessments are performed bilaterally at the time points specified in Table 1. 
Visual function will be evaluated using a methodology appropriate for the age and development 
status of the child, including evaluation of fixation (eg, central, steady and maintained) and fixing 
and following a 5-cm toy. 
Monocular and binocular evaluation of visual function has to be performed. 
If the patient is not able to cooperate with the above methods, another suitable method (eg, Visual 
evoked potentials) can be used to evaluate visual function. 
Cycloplegic refraction will be measured with retinoscopy in each eye and reported separately for 
each eye. 
Ocular motility tests of both eyes (binocular testing) will be assessed to investigate the integrity of 
the extrinsic ocular muscles and their nerves (eg, Cover test or Hirschberg test). 
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9.2.3. 
Safety Procedures 
9.2.3.1. 
Ophthalmic Examinations 
Assessments of ocular safety will include ophthalmologic assessment of the anterior and posterior 
segment (eg, by indirect ophthalmoscopy), and measurement of intraocular pressure (IOP), only 
in patients receiving aflibercept, at the time points specified in Table 1. 
All ophthalmic examinations are to be conducted in both eyes, unless otherwise indicated. 
Intraocular pressure will be measured only in patients receiving aflibercept using a portable 
tonometer (eg, Tono-Pen, Perkin’s Tonometer, or other locally approved device).  The same 
method of IOP measurement should be used in each patient throughout the study.  Intraocular 
pressure will be measured in both eyes prior to the injection, and only in the study eye(s) 
post-injection.  A local anesthetic may be topically applied to the eye(s) being tested (eg, 1 drop 
of oxybuprocain). 
9.2.3.2. 
Physical Examination 
A routine physical examination will assess cardiovascular, respiratory, gastrointestinal, and 
neurological systems and will follow the standard practice of the site.  The assessment will be 
based on the clinical judgment of the physician and aim to evaluate the overall health of the baby.  
Weight, body length, and head circumference will be measure as specified in Table 1. 
9.2.3.3. 
Vital Signs 
Temperature, heart rate, respiratory rate, and blood pressure (BP) will be measured according to 
the local medical practice and regulations, at the time points specified in Table 1.  Study staff 
trained in the assessment of infants will perform these assessments.  Blood pressure and heart rate 
should be assessed with a completely automated device, appropriate for use in infants.  Blood 
pressure must be measured before any study treatment is administered and before PK sampling, if 
applicable. 
Clinically significant abnormal findings will be reported as AEs in the eCRF. 
9.2.3.4. 
Hearing Test 
The preferred hearing test will be a brainstem auditory evoked response (BAER), also referred to 
as an auditory brainstem response (ABR), using electrodes placed on the child’s head and ears to 
generate a response.  The choice of tool will consider the child's age, degree of cooperation, and 
available resources. The child also should be comfortable with the testing situation.  Hearing will 
be assessed once, as specified in Table 1. 
9.2.3.5. 
Laboratory Testing 
The investigator must review the laboratory report, document this review, and record any clinically 
relevant changes occurring during the study in the AE section of the eCRF.  The laboratory reports 
must be filed with the source documents.  Clinically significant abnormal laboratory findings are 
those that are not associated with the underlying disease, unless judged by the investigator to be 
more severe than expected for the patient’s condition. 
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All laboratory tests with values considered clinically significantly abnormal during participation 
in the study or within 4 weeks after the last dose of study intervention should be repeated until the 
values return to normal or baseline or are no longer considered clinically significant by the 
investigator. 
 If such values do not return to normal/baseline within a period of time judged 
reasonable by the investigator, the etiology should be identified and the sponsor 
notified 
 If laboratory values from non-protocol specified laboratory assessments performed at 
the institution’s local laboratory require a change in patient management or are 
considered clinically significant by the investigator (eg, SAE or AE or dose 
modification), then the results must be recorded in the eCRF. 
Laboratory (hematology, chemistry, and urinalysis) analyses will be performed and reviewed at 
screening (visit 1).  A sample is not needed if results from laboratory tests within 8 days prior to 
screening are available and there was no change in the clinical situation from the time of the sample 
to the screening visit. 
In order to avoid discomfort and reduce the probability of urinary tract infections, collection bags 
will be used to collect urine samples if possible. 
Additional samples, including ADA samples, may be collected at any time during the study as 
determined necessary by the investigator or required by local regulations.  This might be true for 
patients receiving rescue treatment with aflibercept. 
Samples for laboratory testing will be collected at visits according to Table 1.  Tests will include: 
Blood Chemistry 
Sodium 
Total protein, serum 
Total bilirubin 
Potassium 
Creatinine 
Uric acid 
Chloride 
Blood urea nitrogen (BUN) 
Creatine phosphokinase (CPK) 
Carbon dioxide 
Aspartate aminotransferase (AST) 
 
Calcium 
Alanine aminotransferase (ALT) 
 
Glucose 
Alkaline phosphatase 
 
Albumin 
Lactate dehydrogenase (LDH) 
 
Hematology 
Hemoglobin 
Differential: 
Hematocrit 
 
Neutrophils 
Red blood cells (RBCs) 
 
Lymphocytes 
White blood cells (WBCs) 
 
Monocytes 
Red cell indices 
 
Basophils 
Platelet count 
 
Eosinophils 
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Urinalysis 
Color 
Glucose 
RBC 
Clarity 
Blood 
Hyaline and other casts 
pH 
Bilirubin 
Bacteria 
Specific gravity 
Leukocyte esterase 
Epithelial cells 
Ketones 
Nitrite 
Crystals 
Protein 
WBC 
Yeast 
 
Abnormal Laboratory Values and Laboratory Adverse Events 
All laboratory values must be reviewed by the investigator or authorized designee. 
Significantly abnormal test results that occur after start of treatment must be repeated to confirm 
the nature and degree of the abnormality.  When necessary, appropriate ancillary investigations 
should be initiated.  If the abnormality fails to resolve or cannot be explained by events or 
conditions unrelated to the study medication or its administration, the Medical/Study Director must 
be consulted. 
The clinical significance of an abnormal test value, within the context of the disease under study, 
must be determined by the investigator. 
Criteria for reporting laboratory values as an AE are provided in Section 10.1.1. 
9.2.3.6. 
Central Nervous System Imaging 
Central nervous system ultrasound examinations will be performed by a well-experienced 
examiner, at the time points specified in Table 1.  The minimum source documentation will include 
electronic or paper documentation (medical report).  Other modalities of imaging (eg, magnetic 
resonance imaging) can be used instead of ultrasound if the alternative test was planned 
independently of the study. 
9.2.4. 
Treatment of Overdose 
For this study, any instance where the investigator assumes that a single IVT dose of more than 
0.4 mg (0.01 mL) was administered will be considered an overdose.  Overdosing with increased 
injection volume may increase IOP.  In these cases, evaluation of IOP and central retinal artery 
perfusion should be performed immediately after the injection and monitored until normalized.  If 
there is severe elevation of IOP causing disruption of central retinal artery perfusion, immediate 
measures to reduce IOP and restore central retinal artery perfusion (eg, performance of an anterior 
chamber paracentesis) should be considered. 
Additionally, after ensuring that IOP and central retinal artery perfusion are in a safe range, the 
investigator should: 
1. Contact the Medical Monitor immediately 
2. Closely monitor the patient for any further AE/SAE and laboratory abnormalities 
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3. Obtain a plasma sample for PK analysis if requested by the Medical Monitor (determined 
on a case-by-case basis) 
4. Document the quantity of the excess administered overdose in the eCRF 
Decisions regarding dose interruptions or modifications will be made by the investigator in 
consultation with the Medical Monitor based on the clinical evaluation of the patient. 
9.2.5. 
Drug Concentration and Measurements 
Since aflibercept levels are systemically demonstrable after IVT administration, systemic 
concentrations of study drug will be collected and described.  The PK evaluations will support the 
safety evaluation.  The objectives of the PK evaluations are to: 
 Describe the individual concentrations of free and bound aflibercept at several time 
points after the first IVT injection up to 4 weeks thereafter 
 Explore the potential influence of demographic and other factors on systemic 
aflibercept concentrations 
 Explore the relationship of systemic exposure and blood pressure 
PK sampling schedule (only applicable for patients treated with aflibercept at baseline) 
One blood sample each for plasma concentrations of study drug will be taken according to the time 
points in Table 1 after BP is measured. 
If the second eye needs to be treated with aflibercept on a day with PK sample collection, the PK 
sample should be collected first. 
To ensure accuracy of the PK analyses, it is critical to accurately record the exact date and time 
(24-hour clock) of all blood samples taken on the eCRF, as well as the exact time of the study 
intervention administration. 
Details about the collection, processing, storage and shipment of samples will be provided 
separately in the laboratory manual.  No capillary blood is allowed for PK samples. 
Additional samples may be collected at any time during the study as determined necessary by the 
investigator or required by local regulations. 
9.2.6. 
Immunogenicity Measurements and Samples 
Samples for ADA assessments will be collected at time points listed in Table 1. 
9.2.7. 
Biomarker Procedures 
Blood pressure as a safety biomarker will be measured using an age-appropriate monitor at the 
time points shown in Table 1.  When applicable, measurements will be taken before study 
treatment, and before PK sampling. 
Only a very limited amount of blood is collected; leftovers (if any) may be used for additional 
research on study drug and/or disease.  Blood volume will not be increased for these research 
purposes. 
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In addition to the biomarkers described above, further biomarkers related to the mode of action or 
the safety of aflibercept and similar drugs may be examined.  The same applies to further 
biomarkers deemed relevant to diseases of the eye and associated health problems.  These 
investigations may include eg, diagnostic, safety, pharmacodynamics, monitoring, or potentially 
predictive biomarkers. 
10. 
SAFETY EVALUATION AND REPORTING 
10.1. 
Recording and Reporting Adverse Events 
10.1.1. 
General Guidelines 
The investigator must promptly record all clinical events occurring during the study data collection 
period (see Section 11.4.5.1) from the time of signing the ICF or the first dose, to the end of the 
treatment period.  Medical conditions that existed or were diagnosed prior to the signing of the 
Informed Consent will be recorded as part of medical history.  Abnormal laboratory values and 
vital signs observed at the time of Informed Consent should also be recorded as medical history.  
Any subsequent worsening (ie, any clinically significant change in frequency and/or intensity) of 
a pre-existing condition that is temporally associated with the use of the study drug should also be 
recorded as an AE. 
At each visit, the investigator will determine whether any AEs have occurred by evaluating the 
patient.  Adverse events may be directly observed, reported spontaneously by the patient's 
parent/legal guardian, or by questioning the patient's parent/legal guardian at each study visit.  
Patient's parent/legal guardian should be questioned in a general way, without asking about the 
occurrence of any specific symptoms.  The Investigator must assess all AEs to determine 
seriousness, severity, and causality, in accordance with the definitions in Section 10.2.  The 
Investigator’s assessment must be clearly documented in the site’s source documentation with the 
Investigator’s signature.  The Investigator should follow up on SAEs (and AESIs) until they have 
resolved or are considered clinically stable; AEs should be followed until they are resolved or last 
study visit, whichever comes first. 
Always report the diagnosis as the AE or SAE term.  When a diagnosis is unavailable, report the 
primary sign or symptom as the AE or SAE term with additional details included in the narrative 
until the diagnosis becomes available.  If the signs and symptoms are distinct and do not suggest 
a common diagnosis, report them as individual entries of AE or SAE.   
Laboratory results, vital signs, and other diagnostic results or findings should be appraised by the 
Investigator to determine their clinical significance.  Isolated abnormal laboratory results, vital 
sign findings, or other diagnostic findings (ie, not part of a reported diagnosis) should be reported 
as AEs if they are symptomatic, lead to study drug discontinuation, dose reduction, require 
corrective treatment, or constitute an AE in the investigator’s clinical judgment. 
For events that are serious due to hospitalization, the reason for hospitalization must be reported 
as the serious adverse event (diagnosis or symptom requiring hospitalization).  A procedure is not 
an AE or SAE, but the reason for the procedure may be an AE or SAE.  Pre-planned (prior to 
signing the Informed Consent Form) procedures, treatments requiring hospitalization for 
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pre-existing conditions that do not worsen in severity, and admission for palliative or social care 
should not be reported as SAEs (see Section 10.2 for Definitions). 
For deaths, the underlying or immediate cause of death should always be reported as an SAE. 
Any SAE that may occur subsequent to the reporting period (end of the treatment period) that the 
Investigator assesses as related to study drug should also be reported. 
All AEs and SAEs are to be reported according to the procedures in Section 10.1.3. 
10.1.2. 
Reporting Procedure  
All events (serious and non-serious) must be reported with investigator’s assessment of the event’s 
seriousness, severity, and causality to the (when applicable: blinded) study drug.  For SAEs, a 
detailed narrative summarizing the course of the event, including its evaluation, treatment, and 
outcome should be provided on the AE CRF.  Specific or estimated dates of event onset, treatment, 
and resolution should be included, when available.  Medical history, concomitant medications, and 
laboratory data that are relevant to the event should also be summarized in the narrative.  For fatal 
events, the narrative should state whether an autopsy was or will be performed, and include the 
results if available.  Information not available at the time of the initial report must be documented 
in a follow-up report.  Source documents (including hospital or medical records, diagnostic reports, 
etc.) will be summarized in the narrative on the AE CRF, and retained at the study center and 
available upon request. 
Urgent safety queries must be followed up and addressed promptly.  Follow-up information and 
response to non-urgent safety queries should be combined for reporting to provide the most 
complete data possible within each follow-up.  
10.1.3. 
Events that Require Expedited Reporting to Sponsor 
The following events also require reporting to the sponsor (or designee) within 24 hours of learning 
of the event: 
 SAEs 
 AESIs:  No AESIs have been defined for this study 
10.2. 
Definitions 
10.2.1. 
Adverse Event 
An AE is any untoward medical occurrence in a patient administered a study drug which may or 
may not have a causal relationship with the study drug.  Therefore, an AE is any unfavorable and 
unintended sign (including abnormal laboratory finding), symptom, or disease which is temporally 
associated with the use of a study drug, whether or not considered related to the study drug (ICH 
E2A Guideline.  Clinical Safety Data Management: Definitions and Standards for Expedited 
Reporting, Oct 1994). 
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10.2.2. 
Serious Adverse Event 
An SAE is any untoward medical occurrence that at any dose: 
 Results in death – includes all deaths, even those that appear to be completely unrelated 
to study drug (eg, a car accident in which a patient is a passenger). 
 Is life-threatening – in the view of the investigator, the patient is at immediate risk of 
death at the time of the event.  This does not include an AE that had it occurred in a 
more severe form, might have caused death. 
 Requires in-patient hospitalization or prolongation of existing hospitalization.  In-
patient hospitalization is defined as a hospital admission (any duration) or an 
emergency room visit for longer than 24 hours.  Prolongation of existing hospitalization 
is defined as a hospital stay that is longer than was originally anticipated for the event, 
or is prolonged due to the development of a new AE as determined by the investigator 
or treating physician. 
 Results in persistent or significant disability/incapacity (substantial disruption of 
one’s ability to conduct normal life functions).   
 Is a congenital anomaly/birth defect 
 Is an important medical event - Important medical events may not be immediately 
life-threatening or result in death or hospitalization, but may jeopardize the patient or 
may require intervention to prevent one of the other serious outcomes listed above (eg, 
intensive treatment in an emergency room or at home for allergic bronchospasm; blood 
dyscrasias or convulsions that do not result in hospitalization; or development of drug 
dependency or drug abuse). 
Criteria for Serious Sight-Threatening Ocular Adverse Events 
Criteria for serious sight-threatening ocular AEs include the following: 
 AE causes a decrease in VA to the level of light perception or worse. 
 AE requires surgical intervention (eg, vitreous tap or biopsy with IVT injection of anti-
infectives, laser or retinal cryopexy with gas) to prevent permanent loss of sight. 
 AE is associated with severe intraocular inflammation (ie, 4 + anterior chamber 
cell/flare or 4 + vitritis) 
 In the opinion of the investigator, AE may require medical intervention to prevent 
permanent loss of sight 
Criteria for reporting SAEs must be followed for these events. 
10.2.3. 
Severity 
The severity of AEs will be graded according to the following scale: 
Mild:  Does not interfere in a significant manner with the patient’s normal functioning level.  It 
may be an annoyance.  Prescription drugs are not ordinarily needed for relief of symptoms, but 
may be given because of personality of the patient. 
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Moderate:  Produces some impairment of functioning but is not hazardous to health.  It is 
uncomfortable or an embarrassment.  Treatment for symptom may be needed. 
Severe:  Produces significant impairment of functioning or incapacitation and is a definite hazard 
to the patient’s health.  Treatment for the symptom may be given and/or the patient may be 
hospitalized. 
If a laboratory value is considered an AE, its severity should be based on the degree of 
physiological impairment the value indicates. 
10.2.4. 
Causality 
The investigator must provide causality assessment as whether or not there is a reasonable 
possibility that the drug caused the adverse event, based on evidence or facts, his/her clinical 
judgment, and the following definitions.  The causality assessment must be made based on the 
available information and can be updated as new information becomes available. 
Include the following when applicable: 
The following factors should be considered when assessing causality: 
 Temporal relationship: time to onset vs time drug was administered 
 Nature of the reactions: immediate vs. long term 
 Clinical and pathological features of the events  
 Existing information about the drug & same class of drugs  
 Concomitant medications  
 Underlying and concurrent illnesses  
 Response to dechallenge (drug discontinuation) or dose reduction  
 Response to rechallenge (re-introduction of the drug) or dose increase, when applicable  
 Patient’s medical and social history  
Causality to the study drug (including study drug administration): 
 Related: 
 The AE follows a reasonable temporal sequence from study drug administration, 
and cannot be reasonably explained by the nature of the reaction, patient’s clinical 
(eg, disease under study, concurrent diseases, concomitant medications), or other 
external factors. 
or 
 The AE follows a reasonable temporal sequence from study drug administration, 
and is a known reaction to the drug under study or its class of drugs, or is predicted 
by known pharmacology. 
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 Not Related: 
 The AE does not follow a reasonable sequence from study drug administration, or 
can be reasonably explained by the nature of the reaction, patient’s clinical state 
(eg, disease under study, concurrent diseases, and concomitant medications) or 
other external factors. 
Causality to the study conduct (protocol specified procedure): 
 Related: 
 The AE follows a reasonable temporal sequence from a protocol specified 
procedure, and cannot be reasonably explained by the nature of the reaction, 
patient’s clinical (eg, disease under study, concurrent diseases, concomitant 
medications), or other external factors. 
 Not Related: 
 The AE does not follow a reasonable sequence from a protocol specified procedure, 
or can be reasonably explained by the nature of the reaction, patient’s clinical state 
(eg, disease under study, concurrent diseases, and concomitant medications) or 
other external factors. 
10.3. 
Safety Monitoring 
The investigator will monitor the safety of study patients at his/her site(s) as per the requirements 
of this protocol and consistent with current Good Clinical Practice (GCP).  Any questions or 
concerns should be discussed with the sponsor in a timely fashion.  The sponsor will monitor the 
safety data from across all study sites.  The Medical/Study Director will have primary 
responsibility for the emerging safety profile of the compound, but will be supported by other 
departments (eg, Pharmacovigilance; Risk Management; Biostatistics and Data Management).  
Safety monitoring will be performed on an ongoing basis (eg, individual review of SAEs) and on 
a periodic cumulative aggregate basis. 
10.4. 
Notifying Health Authorities, Institutional Review Board /Ethics 
Committee, and Investigators 
During the study, the sponsor and/or the CRO will inform health authorities, IECs/IRBs, and the 
participating investigators of any SUSARs (Suspected Unexpected Serious Adverse Reactions) 
occurring in other study centers or other studies of aflibercept, as appropriate per local reporting 
requirements. In addition, the sponsor and/or CRO will comply with any additional local safety 
reporting requirements.  
Upon receipt of the sponsor’s notification of a SUSAR that occurred with the study drug, the 
investigator will inform the Institutional Review Board (IRB)/Ethics Committee (EC) unless 
delegated to the sponsor. 
Event expectedness for aflibercept is assessed against the Reference Safety Information for ROP 
in the current Investigator’s Brochure that is effective for expedited safety reporting. 
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At the completion of the study, the sponsor will report all safety observations made during the 
conduct of the trial in the Clinical Study Report to health authorities and ECs/IRB as appropriate. 
11. 
STATISTICAL PLAN 
This section provides the basis for the statistical analysis plan (SAP) for the study.  The SAP will 
be finalized prior to the end of the study.  The final SAP will be issued before the database lock. 
11.1. 
Statistical Hypothesis 
This study will examine the following hypothesis for the primary efficacy variable regarding the 
proportion of patients with absence of active ROP and unfavorable structural outcomes at week 52 
of chronological age.  Statistical testing will be conducted to demonstrate the non-inferiority of 
the aflibercept group to the laser group, with a non-inferiority margin of 5% and a significance 
level of 5% (one-sided test). 
H0:  pt ≤ pc - 5% versus H1:  pt ≥ pc - 5% 
where pt is the true proportion of patients with absence of active ROP and unfavorable structural 
outcomes at week 52 of chronological age for the aflibercept group, and pc is the true proportion 
of patients with absence of active ROP and unfavorable structural outcomes at week 52 of 
chronological age for the laser group. 
The proposed non-inferiority margin of 5% is smaller than the smallest difference between laser 
and ranibizumab in RAINBOW (a phase 3 study comparing the effect of IVT ranibizumab to laser 
in the management of ROP), and not greater than the difference between the two ranibizumab 
doses. 
11.2. 
Justification of Sample Size 
From the RAINBOW study, the response rate was 66.1% for the laser group and 88.1% for the 
0.2 mg ranibizumab group in zone II disease. Furthermore, based on the clinical evidence for the 
aflibercept investigator-initiated studies (Salman, 2015) (Huang, 2018) (Sukgen, 2018) 
(Sidorenko, publication in progress), the response rates range all the way up to 100% (with at least 
2 studies demonstrating a 100% response rate in terms of favorable anatomic structural 
outcomes/prevention of unfavorable anatomic structural outcomes) with IVT aflibercept doses 
ranging from 0.4 mg to 1 mg.  An estimated 90% response rate for the aflibercept group and 66.1% 
response rate for the laser group would be a safe assumption.  It is assumed that the proportion of 
patients with absence of active ROP will be in line with this. 
A sample size of 84 patients in the aflibercept group and 28 patients in the laser group (randomized 
in a 3:1 ratio) will provide 92% power for rejecting the null hypothesis at a 1-sided 5% significance 
level. 
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11.3. 
Analysis Sets 
11.3.1. 
Full Analysis Set 
The full analysis set (FAS) will include all randomized patients who received any study treatment.  
The analysis on the FAS will be performed according to the treatment assigned at baseline (as 
randomized).  
11.3.2. 
Per Protocol Set 
The per protocol set (PPS) includes all patients in the FAS who had no important protocol 
deviations.  Final determinations of the PPS will be made before database lock.  Analysis of the 
PPS will be performed according to the treatment actually received (as treated).  The PPS will be 
used for sensitivity analysis of the primary endpoint. 
11.3.3. 
Safety Analysis Set 
The safety analysis set (SAF) includes all randomized patients who received any study treatment 
(active or laser); it is based on the treatment actually received (as treated).  Treatment 
compliance/administration and all clinical safety variables will be analyzed using the SAF. 
11.3.4. 
Pharmacokinetic Analysis Set 
The PK analysis population includes all patients who received any study drug and who had a at 
least 1 non-missing result following the first dose of study drug. 
11.3.5. 
Immunogenicity Analysis Set 
The ADA analysis set includes all patients who received any study drug and had at least 1 non-
missing ADA result following the first dose of study drug. 
11.4. 
Statistical Methods 
Unless stated otherwise, all variables will be analyzed descriptively with appropriate statistical 
methods:  continuous variables by sample statistics (ie, mean, standard deviation, median, 
quartiles, minimum, and maximum), and categorical variables by frequencies and percentages. 
All statistical analyses will be performed using Statistical Analysis System (SAS); the version used 
will be specified in the SAP. 
11.4.1. 
Patient Disposition 
The following will be provided: 
 The total number of screened patients:  met the inclusion criteria regarding the target 
indication and signed the ICF 
 The total number of randomized patients:  received a randomization number 
 The total number of patients who discontinued the study, and the reasons for 
discontinuation 
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 The total number of patients who discontinued from study treatment, and the reasons 
for discontinuation 
 A listing of patients treated but not randomized, patients randomized but not treated, 
and patients randomized but not treated as randomized 
 A listing of patients prematurely discontinued from treatment, along with reasons for 
discontinuation 
11.4.2. 
Demography and Baseline Characteristics 
Demographic and baseline characteristics will be summarized descriptively by treatment group, 
and by all patients combined. 
11.4.3. 
Efficacy Analyses 
11.4.3.1. Primary Efficacy Analysis 
The primary efficacy variable analysis will be conducted on the FAS.  The primary analysis is a 
statistical evaluation of non-inferiority of aflibercept vs. laser at week 52 of chronological age, 
with respect to the primary efficacy variable (the proportion of patients with absence of active 
ROP and of unfavorable structural outcomes).  The non-inferiority margin is set at 5%.  Superiority 
testing will follow if non-inferiority is established.  Sensitivity analysis of the primary endpoint 
will also be conducted based on the PPS. 
If data are available from the week 40 of chronological age visit, it will be carried forward (LOCF) 
to the EOS visit for analysis.  If the patient discontinued at or before this visit, no data will be 
carried forward and the patient will be considered a non-responder.  For patients with both eyes 
enrolled in the study, both eyes must meet the endpoint.  Patients with only one study eye enrolled 
will be responders if the respective eye responds. 
The statistical analysis will be performed using the Cochran-Mantel-Haenszel method stratified 
by baseline ROP status.  The 1-sided 95% Mantel-Haenszel confidence interval using normal 
approximation of the difference of response rates between the aflibercept group and the laser group 
will be calculated.  Aflibercept will be considered to be non-inferior to laser if the confidence 
interval of the difference lies entirely above -5%. 
Furthermore, eyes will be considered to be non-responders if rescue treatment is given.  The 
primary analysis for this endpoint will be based on the investigator assessment of, and sensitivity 
analyses based on the central RC data will be conducted. 
11.4.3.2. Secondary and Exploratory Efficacy Analyses 
The efficacy variables will be analyzed using the same method as for the analysis of the primary 
efficacy variable. 
11.4.4. 
Control of Multiplicity 
If the primary endpoint is declared significant, a hierarchical testing procedure will be used for the 
analysis of the secondary endpoints to control the overall alpha error rate at the 0.05 level.  The 
order of the endpoints for hierarchical testing will be specified in the SAP. 
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11.4.5. 
Safety Analysis 
Safety variables will be summarized on the SAF through week 52 of chronological age for both 
treatment groups. 
11.4.5.1. Adverse Events 
Definitions 
For safety variables, 3 observation periods are defined: 
 The pretreatment period is defined as the time from signing the ICF to before the first 
dose of study drug. 
 The treatment period is defined as the day from first dose of study drug to the last dose 
of study drug. 
 The post-treatment period is defined as the time after the last dose of study drug. 
A TEAE is defined as an event (or an exacerbation of a preexisting event during the treatment 
period) that is observed or reported after the first administration of study drug, and no later than 
30 days after last administration of study drug. 
Analysis 
All AEs reported in this study will be coded using the currently available version of the Medical 
Dictionary for Regulatory Activities (MedDRA®).  Coding will be to lowest level terms.  The 
verbatim text, the preferred term (PT), and the primary system organ class (SOC) will be listed. 
Summaries of all TEAEs by treatment group, and by ocular study eye, ocular fellow eye, and 
non-ocular TEAEs, will include: 
 The number (n) and percentage (%) of patients with at least 1 TEAE by SOC and PT 
 TEAEs by severity (according to the grading scale outlined in Section 10.2.3), 
presented by SOC and PT 
 TEAEs by relationship to drug or injection procedures (related, not related), presented 
by SOC and PT 
Deaths and other SAEs will be listed and summarized by treatment group. 
Treatment-emergent adverse events leading to permanent treatment discontinuation will be listed 
and summarized by treatment group. 
11.4.5.2. Ocular Safety 
Ocular safety variables (eg, IOP measurements) will be analyzed descriptively at their scheduled 
visit, including changes from baseline.  All ocular safety parameters will be summarized using 
descriptive statistics.  Information from fundus photography and FA (including the substudy) may 
also be used to assess ocular safety. 
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11.4.5.3. Other Safety 
Vital Signs 
Vital signs (temperature, pulse, blood pressure, and respiration rate) will be summarized by 
baseline and change from baseline to each scheduled assessment time with descriptive statistics. 
Laboratory Tests 
Laboratory test results will be summarized by baseline and change from baseline to each scheduled 
assessment time with descriptive statistics. 
Number and percentage of patients with a potentially clinically significant value (PCSV) at any 
post-randomization time point will be summarized for each clinical laboratory test for all patients 
and separately for patients in whom the PCSV criterion was normal or missing at baseline.  
Shift tables based on baseline normal/abnormal and other tabular and graphical methods may be 
used to present the results for laboratory tests of interest. 
Listings will be provided with flags indicating the out of laboratory range values. 
11.4.5.4. Treatment Exposure 
Exposure to study drug will be examined for each patient.  The total number of treatments 
administered to each patient and the duration of treatment will be analyzed and summarized using 
descriptive statistics by treatment group in the SAF and FAS populations 
11.4.5.5. Treatment Compliance 
Compliance with protocol defined study medication will be calculated as follows: 
Treatment compliance = (number of received injections through a given week)/(number of planned 
injections during the period of participation in the study through the given week) x 100%. 
11.4.6. 
Pharmacokinetics 
11.4.6.1. Analysis of Drug Concentration Data 
The plasma concentrations of free and bound aflibercept over time will be summarized by 
descriptive statistics. 
No formal statistical hypothesis testing will be performed.  
11.4.6.2. Pharmacokinetics and Pharmacokinetics/Pharmacodynamics Analyses 
Population PK and exposure-response analyses for blood pressure may be performed, as 
appropriate, and presented in separate reports. 
11.4.7. 
Analysis of Immunogenicity Data 
Anti-drug antibody status (negative or positive) and titer over the study duration may be classified 
as follows:  
 Treatment-emergent ADA response, defined as any post-dose positive ADA assay 
response when the baseline results are negative 
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 Treatment-boosted ADA response, defined as any post-dose positive ADA assay 
response that is 4-fold over baseline titer levels when baseline is positive in the ADA 
assay  
 Maximum ADA titer values 
 Low (titer <1,000) 
 Moderate (1,000≤ titer ≤10,000) 
 High (titer >10,000)  
 NAb status (positive or negative) for samples that are positive in the ADA assay 
Listings of ADA positivity and titers presented by patient, time point, and study treatment received 
will be provided.  Incidence of treatment-emergent and treatment-boosted ADA will be assessed 
as absolute occurrence (N) and percent of patients (%), grouped by study treatment received. 
The influence of ADAs on individual PK profiles will be evaluated.  Assessment of impact of 
ADA on safety and efficacy may be provided. 
11.4.8. 
Analysis of Biomarker Data 
Biomarker exploratory analyses will be described in the SAP. 
11.5. 
Interim Analysis 
No interim analysis is planned. 
11.6. 
Additional Statistical Data Handling Conventions 
The following analysis and data conventions will be followed: 
Definition of baseline: 
 The baseline assessment is defined as the latest valid pre-dose assessment 
General rules for handling missing data: 
 Rules for handling missing data for efficacy assessments are described in the efficacy 
analysis section.  Additional details will be provided in the SAP. 
 AE variables 
For some AEs it is important to determine whether the AE started before or after the 
first active aflibercept injection. If the AE start date is partially missing, it will be 
imputed by the latest possible date (considering other available data, e.g., stop date) to 
be conservative. 
 Prior/concomitant medication 
For the tabulation of prior and concomitant medication, partially missing start dates of 
the medication will be imputed by the earliest possible time point, partially missing 
stop dates will be imputed by the latest possible time point 
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 No imputations for missing laboratory data, vital sign data, or physical examination 
data will be made. 
Unscheduled assessments 
 Assessments taken outside of protocol allowable windows will be displayed according 
to the CRF assessment recorded by the investigator.  Unscheduled assessments will not 
be included in the summaries. 
11.7. 
Statistical Considerations Surrounding the Premature Termination 
of a Study 
If the study is terminated prematurely, only those parameters required for the development 
program and/or reporting to regulatory authorities will be summarized.  Investigator and sponsor 
responsibilities surrounding the premature termination of a study are presented in Section 15.1. 
12. 
QUALITY CONTROL AND QUALITY ASSURANCE 
In accordance with ICH E6, the sponsor is responsible for quality assurance to ensure that the 
study is conducted and the data generated, recorded, and reported in compliance with the protocol, 
GCP, and any applicable regulatory requirement(s).  The planned quality assurance and quality 
control procedures for the study are described in this section. 
12.1. 
Data Management and Electronic Systems 
12.1.1. 
Data Management 
A data management plan specifying all relevant aspects of data processing for the study (including 
data validation [quality-checking], cleaning, correcting, releasing) will be maintained and stored 
at Regeneron (Sponsor). 
A medical coding plan will specify the processes and the dictionary used for coding.  All data 
coding (eg, AEs, baseline findings, medication, medical history/surgical history/ophthalmic 
history) will be done using internationally recognized and accepted dictionaries. 
The CRF data for this study will be collected with an electronic data capture (EDC) system. 
12.1.2. 
Electronic Systems 
Electronic systems that may be used to process and/or collect data in this study will include the 
following: 
 IVRS/IWRS system – randomization, study drug supply 
 EDC system – data capture – Medidata Rave 
 Statistical Analysis System (SAS) – statistical review and analysis 
 Pharmacovigilance safety database 
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12.2. 
Study Monitoring 
12.2.1. 
Monitoring of Study Sites 
The study monitor and/or designee (eg, contract research organization [CRO] monitor) will visit 
each site prior to enrollment of the first patient, and periodically during the study.  This study will 
use the principles of risk-based monitoring (ICH).  This means that the number of visits for any 
given site may vary based on site risk indicators.  The investigator must allow study-related 
monitoring. 
The study monitors will perform ongoing source data review to verify that data recorded in the 
CRF by authorized site personnel are accurate, complete, and verifiable from source documents, 
that the safety and rights of patients are being protected, and that the study is being conducted in 
accordance with the current approved protocol version and any other study agreements, ICH GCP, 
and all applicable regulatory requirements. 
12.2.2. 
Source Document Requirements 
Investigators are required to prepare and maintain adequate and accurate patient records (source 
documents).  The site is responsible to ensure quality within their records and systems and are 
accountable for ensuring that all source data and CRF data are timely, accurate and complete. 
The investigator must keep all source documents on file with the CRF (throughout this protocol, 
CRF refers to either a paper CRF or an electronic CRF).  Case report forms and source documents 
must be available at all times for inspection by authorized representatives of the sponsor and 
regulatory authorities. 
12.2.3. 
Case Report Form Requirements 
Study data obtained in the course of the clinical study will be recorded on electronic Case Report 
Forms (CRFs) within the EDC system by trained site personnel.  All required CRFs must be 
completed for each and every patient enrolled in the study.  The investigator must ensure the 
accuracy, completeness, and timeliness of the data reported to the sponsor in the CRFs.  After 
review of the clinical data for each patient, the investigator must provide an electronic signature.  
A copy of each patient CRF casebook is to be retained by the investigator as part of the study 
record and must be available at all times for inspection by authorized representatives of the sponsor 
and regulatory authorities. 
Corrections to the CRF will be entered in the CRF by the investigator or an authorized designee.  
All changes, including date and person performing corrections, will be available via the audit trail, 
which is part of the EDC system.  For corrections made via data queries, a reason for any alteration 
must be provided. 
12.3. 
Audits and Inspections 
This study may be subject to a quality assurance audit or inspection by the sponsor or regulatory 
authorities.  Should this occur, the investigator is responsible for: 
 Informing the sponsor of a planned inspection by the authorities as soon as notification 
is received, and authorizing the sponsor’s participation in the inspection 
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 Providing access to all necessary facilities, study data, and documents for the inspection 
or audit 
 Communicating any information arising from inspection by the regulatory authorities 
to the sponsor immediately 
 Taking all appropriate measures requested by the sponsor to resolve the problems found 
during the audit or inspection 
Documents subject to audit or inspection include but are not limited to all source documents, CRFs, 
medical records, correspondence, ICFs, IRB/EC files, documentation of certification and quality 
control of supporting laboratories, and records relevant to the study maintained in any supporting 
pharmacy facilities.  Conditions of study material storage are also subject to inspection.  In 
addition, representatives of the sponsor may observe the conduct of any aspect of the clinical study 
or its supporting activities both within and outside of the investigator's institution. 
In all instances, the confidentiality of the data must be respected. 
12.4. 
Study Documentation 
12.4.1. 
Certification of Accuracy of Data 
A declaration assuring the accuracy and content of the data recorded on the CRF/eCRF must be 
signed electronically by the investigator.  This signed declaration accompanies each set of patient 
final CRFs/eCRFs that will be provided to the sponsor. 
12.4.2. 
Retention of Records 
The investigator must retain all essential study documents, including ICFs, source documents, 
investigator copies of CRFs, and drug accountability records for at least 15 years following the 
completion or discontinuation of the study, or longer, if a longer period is required by relevant 
regulatory authorities.  The investigator must obtain written approval from the sponsor before 
discarding or destroying any essential study documents during the retention period following study 
completion or discontinuation.  Records must be destroyed in a manner that ensures 
confidentiality. 
If the investigator's personal situation is such that archiving can no longer be ensured, the 
investigator must inform the sponsor (written notification) and the relevant records will be 
transferred to a mutually agreed-upon destination.  
13. 
ETHICAL AND REGULATORY CONSIDERATIONS  
13.1. 
Good Clinical Practice Statement 
It is the responsibility of both the sponsor and the investigator(s) to ensure that this clinical study 
will be conducted in accordance with the ethical principles that have their origin in the Declaration 
of Helsinki, and that are consistent with the ICH guidelines for GCP and applicable regulatory 
requirements. 
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13.2. 
Informed Consent 
The principles of informed consent are described in ICH guidelines for Good Clinical Practice. 
The ICF used by the investigator must be reviewed and approved by the sponsor prior to 
submission to the appropriate IRB/EC.  A copy of the IRB/EC -approved ICF and documentation 
of approval must be provided to the sponsor before study drug will be shipped to the study site. 
It is the responsibility of the investigator or designee (if acceptable by local regulations) to obtain 
written informed consent from each patient's parent(s) or legal guardian(s) prior to the patient’s 
participation in the study and after the aims, methods, objectives, and potential hazards of the study 
have been explained to the fullest possible extent in language that the patient's parent(s) or legal 
guardian(s) can understand.  The ICF should be signed and dated by the patient’s parent(s) or legal 
guardian(s) and the same investigator or designee who explained the ICF. 
Local law must be observed in deciding whether 1 or both parents/guardians consent is required. 
If the patient’s parent(s) or legal guardian(s) can write but cannot read, the ICF will be read to 
them before signing their name on the ICF.  If the patient’s parent(s) or legal guardian(s) can 
understand but can neither write nor read, the ICF will be read to them in the presence of an 
impartial witness, who will sign and date the ICF to confirm that informed consent was given. 
If only 1 parent or guardian signs the consent form, the investigator must document the reason the 
other parent or guardian did not sign.  The patient's parent/legal guardian may also be required to 
sign and date the ICF, as determined by the IRB/EC and in accordance with the local regulations 
and requirements. 
 Patient's parent(s)/legal guardian(s) who can write but cannot read will have the assent 
form read to them before writing their name on the form. 
 Patient's parent(s)/legal guardian(s) who can understand but who can neither write nor 
read will have the ICF read to them in presence of an impartial witness, who will sign 
and date the ICF to confirm that informed consent was given. 
The original ICF must be retained by the investigator as part of the patient’s study record, and a 
copy of the signed ICF must be given to the patient’s parent(s) or legal guardian(s). 
If new safety information results in significant changes in the risk/benefit assessment, or if there 
are significant changes to the study procedures, the ICF must be reviewed and updated 
appropriately.  All study patients’ parent(s) or legal guardian(s) must be informed of the new 
information and provide their written consent if they wish the patient to continue in the study.  The 
original signed revised ICF must be maintained in the patient’s study record and a copy must be 
given to the patient’s parent(s) or legal guardian(s). 
13.3. 
Patients’ Confidentiality and Data Protection 
The investigator must take all appropriate measures to ensure that the anonymity of each study 
patient will be maintained.  Patients should be identified by a patient identification number and 
date of birth, on CRFs or other documents submitted to the sponsor. Documents that will not be 
submitted to the sponsor (eg, signed ICF) must be kept in strict confidence. 
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The patient’s and investigator's personal data, which may be included in the sponsor database, will 
be treated in compliance with all applicable laws and regulations.  The sponsor shall take all 
appropriate measures to safeguard and prevent access to this data by any unauthorized third party. 
13.4. 
Institutional Review Board/Ethics Committee 
An appropriately constituted IRB/EC, as described in ICH guidelines for GCP, must review and 
approve: 
 The protocol, ICF, and any other materials to be provided to the patient's parent(s)/legal 
guardian(s) (eg, advertising) before any patient may be enrolled in the study 
 Any amendment or modification to the study protocol or ICF before implementation, 
unless the change is necessary to eliminate an immediate hazard to the patient, in which 
case the IRB/EC should be informed as soon as possible  
 Ongoing studies on an annual basis or at intervals appropriate to the degree of risk 
In addition, the IRB/EC should be informed of any event likely to affect the safety of patients or 
the continued conduct of the clinical study. 
A copy of the IRB/EC approval letter with a current list of the IRB/EC members and their functions 
must be received by the sponsor prior to shipment of drug supplies to the investigator.  The 
approval letter should include the study number and title, the documents reviewed, and the date of 
the review. 
Records of the IRB/EC review and approval of all study documents (including approval of ongoing 
studies) must be kept on file by the investigator. 
13.5. 
Clinical Study Data Transparency 
Final study results will be published on a public clinical trial website according to applicable local 
guidelines and regulations.  Treatment codes will be disseminated to each investigation site 
thereafter. 
14. 
PROTOCOL AMENDMENTS 
The sponsor may not implement a change in the design of the protocol or ICF without an IRB/EC-
approved amendment.  Where required per local legislation, regulatory authority approval will also 
be sought. 
15. 
PREMATURE TERMINATION OF THE STUDY OR CLOSEOUT 
OF A SITE 
15.1. 
Premature Termination of the Study 
The sponsor has the right to terminate the study prematurely.  Reasons may include efficacy, 
safety, or futility, among others.  Should the sponsor decide to terminate the study, the 
investigator(s) will be notified in writing. 
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15.2. 
Closeout of a Site 
The sponsor and the investigator have the right to closeout a site prematurely. 
Investigator’s Decision 
The investigator must notify the sponsor of a desire to closeout a site in writing, providing at least 
30 days’ notice.  The final decision should be made through mutual agreement with the sponsor.  
Both parties will arrange the closeout procedures after review and consultation. 
Sponsor’s Decision 
The sponsor will notify the investigator(s) of a decision to closeout a study site in writing.  Reasons 
may include the following, among others: 
 The investigator has received all items and information necessary to perform the study, 
but has not enrolled any patient within a reasonable period of time 
 The investigator has violated any fundamental obligation in the study agreement, 
including but not limited to, breach of this protocol (and any applicable amendments), 
breach of the applicable laws and regulations, or breach of any applicable ICH 
guidelines  
 The total number of patients required for the study are enrolled earlier than expected 
In all cases, the appropriate IRB/EC and Health Authorities must be informed according to 
applicable regulatory requirements, and adequate consideration must be given to the protection of 
the patients’ interests. 
16. 
CONFIDENTIALITY 
Confidentiality of information is provided as a separate agreement. 
17. 
FINANCING AND INSURANCE 
Financing and insurance information is provided as a separate agreement. 
18. 
PUBLICATION POLICY  
Publication rights and procedures will be outlined in a separate clinical study agreement. 
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19. 
REFERENCES 
Blencowe H, Lee AC, Cousens S, et al.  Preterm birth-associated neurodevelopmental impairment 
estimates at regional and global levels for 2010.  Pediatr Res 2013;74 Suppl 1:17-34. 
Gilbert C, Rahi J, Eckstein M, et al.  Retinopathy of prematurity in middle-income countries.  The 
Lancet 1997;350(9070):12-14. 
Harder BC, Schlichtenbrede FC, Von Baltz S, et al.  Intravitreal bevacizumab for retinopathy of 
prematurity: refractive error results.  Am J Ophthalmol 2013 Jun;155(6):1119-1124. 
Hartnett ME.  Vascular endothelial growth factor antagonist therapy for retinopathy of 
prematurity.  Clin Perinatol 2014;41:925-943. 
Hartnett ME, Morrison MA, Smith S, et al.  Genetic variants associated with severe retinopathy of 
prematurity in extremely low birth weight infants.  Invest Ophthalmol Vis Sci 2014 
Oct;55(10):6194-6203. 
Hellström A and Smith LEH. Dammann O. Retinopathy of prematurity. Lancet 
2013;382(9902):1445-1457. 
Huang CY, Lien R, Wang NK, et al.  Changes in systemic vascular endothelial growth factor levels 
after intravitreal injection of aflibercept in infants with retinopathy of prematurity.  Graefes Arch 
Clin Exp Ophthalmol 2018;256, 479-487.  
Hwang CK, Baker HG, Hutchinson AK, et al.  Outcomes after intravitreal bevacizumab versus 
laser photocoagulation for retinopathy of prematurity: a 5-year retrospective analysis.  
Ophthalmology 2015 May;122(5):1008-1015. 
Mintz-Hittner HA, Kennedy KA, and Chuang AZ.  Efficacy of intravitreal bevacizumab for stage 
3+ retinopathy of prematurity.  NEJM 2011;364(7):603-15.  
Mutlu FM and Sarici SU.  Treatment of retinopathy of prematurity: a review of conventional and 
promising new therapeutic options.  Int J Ophthalmol 2013;6:228-236. 
Painter SL, Wilkinson AR, Desai P, et al.  Incidence and treatment of retinopathy of prematurity 
in England between 1990 and 2011: database study. Br J of Ophth 2015;99(6):807. 
Pertl L, Steinwender G, Mayer C, et al.  A Systematic Review and Meta-Analysis on the Safety of 
Vascular Endothelial Growth Factor (VEGF) Inhibitors for the Treatment of Retinopathy of 
Prematurity.  PLoS One. 2015;10:e0129383. 
Salman AG and Said AM.  Structural, visual and refractive outcomes of intravitreal aflibercept 
injection in high-risk prethreshold type 1 retinopathy of prematurity.  Ophth Res 2015;53:15-20. 
Sidorenko EE.  Treatment of retinopathy of prematurity with aflibercept in Russia.  Presented at: 
17th International Conference on Clinical and Experimental Ophthalmology.  2018 Oct 01-03. 
Moscow, 
Russia. 
Available 
from: 
 
https://ophthalmology.conferenceseries.com/ 
abstract/2018/treatment-of-retinopathy-of-prematurity-with-aflibercept-in-russia  
Sidorenko Pulication in progress, Azuma Oral Communication 
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Stone J, Chan-Ling T, Pe'er J, et al.  Roles of Vascular Endothelial Growth Factor and Astrocyte 
Degeneration in the Genesis of Retinopathy of Prematurity. Invest Ophthalmol Vis Sci 
1996;37:290-99. 
Sukgen EA and Kocluk Y.  Comparison of clinical outcomes of intravitreal ranibizumab and 
aflibercept treatment for retinopathy of prematurity.  Graefe’s Archive for Clinical and 
Experimental 
Ophthalmology. 
 
Revised 
2018 
Apr 
16. 
 
Available 
from: 
https://doi.org/10.1007/s00417-018-4168-5. 
Young TL, Anthony DC, Pierce E, et al. Foley E, Smith LE.  Histopathology and vascular 
endothelial growth factor in untreated and diode laser-treated retinopathy of prematurity.  J 
AAPOS. 1997;1:105-10. 
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20. 
INVESTIGATOR’S AGREEMENT 
I have read the attached protocol:  Randomized, Controlled, Multi-Center Study to Assess the 
Efficacy, Safety, and Tolerability of Intravitreal Aflibercept Compared to Laser Photocoagulation 
in Patients with Retinopathy of Prematurity, and agree to abide by all provisions set forth therein. 
I agree to comply with the current International Council for Harmonisation Guideline for Good 
Clinical Practice and the laws, rules, regulations, and guidelines of the community, country, state, 
or locality relating to the conduct of the clinical study. 
I also agree that persons debarred from conducting or working on clinical studies by any court or 
regulatory agency will not be allowed to conduct or work on studies for the sponsor or a partnership 
in which the sponsor is involved.  I will immediately disclose it in writing to the sponsor if any 
person who is involved in the study is debarred, or if any proceeding for debarment is pending, or, 
to the best of my knowledge, threatened. 
This document contains confidential information of the sponsor, which must not be disclosed to 
anyone other than the recipient study staff and members of the IRB/EC.  I agree to ensure that this 
information will not be used for any purpose other than the evaluation or conduct of the clinical 
investigation without the prior written consent of the sponsor. 
 
 
 
 
 
 
(Signature of Investigator) 
 
(Date) 
 
 
 
(Printed Name) 
 
 
 
 
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APPENDIX 1. 
FLUORESCEIN ANGIOGRAPHY SUB-STUDY 
Fluorescein Angiography Sub-Study 
This is an exploratory analysis for fluorescein angiography (FA) conducted as a sub-study to 
protocol VGFTe-ROP-1920.  Investigational sites that are participating in VGFTe-ROP-1920 may 
choose to participate in this optional, exploratory FA sub-study.  In order to participate, sites must 
have the ability and prior experience to perform FAs on the patient/study population identified as 
part of VGFTe-ROP-1920.  These specifically include experience in performing FA in pre-term 
infants using a digital fiber optic contact FA in patients treated with vascular endothelial growth 
factor inhibitors for retinopathy of prematurity. 
A. 
Sub-study Objective 
The objective of this sub-study is to explore the use of FA to assess vascular leakage, non-
perfusion, ischemia and the development of the normal retinal vasculature in the population under 
study pre- and post-treatment.  It will involve the injection/administration of intravenous/oral 
fluorescein dye into the patient, followed by taking wide-angle photographs and angiographic 
images of the retina in both eyes.  As FA has the ability to image vessels and leakage accurately, 
it may provide additional information related to treatment response and on the disease state beyond 
that achievable with Color Fundus Photography Imaging.  This will also help us generate a Non-
Perfusion Index (NPI).  The NPI is the ratio of non-perfused (i.e. ischemic) retina to perfused retina 
as seen on FA. 
B. 
Number of Patients and Sites 
Patients will be enrolled at the investigator’s discretion at sites that have access to FA imaging.  
There will be no cap on the number of patients or sites that may participate.  
C. 
Timing of Fluorescein Angiography 
FA will be performed prior to administration of very first study treatment (optional yet desired to 
compare NPI change) and at the week 52 of chronological age visit (mandatory) (Table 1).  
Additional FA may be performed at the investigator’s discretion in conjunction with all the other 
scheduled study visits for the main study, as well as at any other treatment intervention. 
Detailed instructions for procedure, image collection and transmission will be provided in the 
study procedure manual. 
 
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SIGNATURE OF SPONSOR’S RESPONSIBLE OFFICERS 
(Medical/Study Director, Regulatory Representative, Clinical Study Lead, and 
Biostatistician) 
To the best of my knowledge, this report accurately describes the planned conduct of the study. 
Study Title: 
 Randomized, Controlled, Multi-Center Study to Assess the Efficacy, Safety, and 
Tolerability of Intravitreal Aflibercept Compared to Laser Photocoagulation in Patients with 
Retinopathy of Prematurity 
Protocol Number: 
VGFTe-ROP-1920 
Protocol Version: 
VGFTe-ROP-1920 Amendment 1 
 
See appended electronic signature page 
Sponsor’s Responsible Medical/Study Director 
 
 
See appended electronic signature page 
Sponsor’s Responsible Regulatory Liaison 
 
 
See appended electronic signature page 
Sponsor’s Responsible Clinical Study Lead 
 
 
See appended electronic signature page 
Sponsor’s Responsible Biostatistician 
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Signature Page for VV-RIM-00083535 v1.0
Signature Page for VV-RIM-00083535 v1.0 Approved
ESig Approval
00
ESig Approval
2019 21:04:37 GMT+0000
ESig Approval
2019 21:13:21 GMT+0000
ESig Approval
9 22:30:51 GMT+0000
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SAP_003.pdf: 
STATISTICAL ANALYSIS PLAN 
CONFIDENTIAL 
Page 1 of 34 
Title: 
RANDOMIZED, CONTROLLED, MULTI-CENTER STUDY 
TO ASSESS THE EFFICACY, SAFETY, AND 
TOLERABILITY OF INTRAVITREAL AFLIBERCEPT 
COMPARED TO LASER PHOTOCOAGULATION IN 
PATIENTS WITH RETINOPATHY OF PREMATURITY   
Protocol Number: 
VGFTe-ROP-1920 Amendment 1 29Jul2019 
Investigational product: 
Intravitreal Aflibercept Injection 
Sponsor: 
Regeneron Pharmaceuticals, Inc. 
Study Biostatistician: 
Clinical Trial Manager: 
Study Medical Director: 
Date: 
05MAY2021 
Version: 
4.0 
VV-RIM-00152939-1.0 Approved - 07 May 2021 GMT-5:00
NCT Number:  NCT04101721
EudraCT Number:  2019-001764-29 
Clinical Development and Regulatory Affairs 
Biostatistics and Data Management 


Regeneron Pharmaceuticals, Inc. 
 
VGFTe-ROP-1920 
Statistical Analysis Plan 
 
05MAY2021 
 
CONFIDENTIAL  
Page 2 of 34 
The approval signatures below indicate that these individuals have reviewed the Statistical 
Analysis Plan (SAP) and agreed on the planned analysis defined in this document for reporting. 
 
 
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TABLE OF CONTENTS 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ....................................................6 
DOCUMENT VERSION HISTORY ..............................................................................................8 
1. 
OVERVIEW .................................................................................................................9 
1.1. 
Background/Rationale ..................................................................................................9 
1.2. 
Study Objectives ...........................................................................................................9 
1.2.1. 
Primary Objectives .......................................................................................................9 
1.2.2. 
Secondary Objectives .................................................................................................10 
1.2.3. 
Exploratory Objectives ...............................................................................................10 
1.2.4. 
Modifications from the Statistical Section in the Final Protocol ................................10 
1.2.5. 
Modifications from the Approved Statistical Analysis Plan ......................................10 
2. 
INVESTIGATION PLAN ..........................................................................................10 
2.1. 
Sample Size and Power Considerations .....................................................................10 
2.2. 
Study Design and Randomization ..............................................................................11 
3. 
ANALYSIS POPULATIONS ....................................................................................13 
3.1. 
Full Analysis Set .........................................................................................................13 
3.2. 
Safety Analysis Set .....................................................................................................13 
3.3. 
Per Protocol Set ..........................................................................................................13 
3.4. 
All Randomized Set ....................................................................................................14 
3.5. 
Pharmacokinetic Analysis Set ....................................................................................14 
3.6. 
Immunogenicity Analysis Sets ...................................................................................14 
4. 
ANALYSIS VARIABLES .........................................................................................14 
4.1. 
Demographic and Baseline Characteristics ................................................................14 
4.2. 
Medical History ..........................................................................................................15 
4.3. 
Pre-Treatment / Concomitant Medication ..................................................................15 
4.4. 
Exposure and Additional Treatment to Study Treatment ...........................................15 
4.5. 
Efficacy Variable ........................................................................................................16 
4.5.1. 
Primary Efficacy Variable (s) .....................................................................................16 
4.5.2. 
Secondary Efficacy Variable(s) ..................................................................................16 
4.5.3. 
Exploratory Efficacy Variable(s) ................................................................................16 
4.6. 
Safety Variables ..........................................................................................................17 
4.6.1. 
Adverse Events and Serious Adverse Events .............................................................17 
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4.6.2. 
Surgeries .....................................................................................................................17 
4.6.3. 
Laboratory Safety Variables .......................................................................................17 
4.6.4. 
Vital Signs ..................................................................................................................18 
4.6.5. 
Ocular Safety Measures ..............................................................................................18 
4.7. 
Pharmacokinetic Variables .........................................................................................18 
4.8. 
Immunogenicity Variables ..........................................................................................18 
5. 
STATISTICAL METHODS .......................................................................................18 
5.1. 
Statistical Hypothesis ..................................................................................................18 
5.2. 
Subject Disposition .....................................................................................................19 
5.3. 
Demographics, Baseline Characteristics, Medical History, and Prior/ 
Concomitant Medication ............................................................................................19 
5.4. 
Extent of Study Treatment Exposure ..........................................................................20 
5.5. 
Analyses of Efficacy Variables ..................................................................................20 
5.5.1. 
Analysis of Primary Efficacy Variable(s) ...................................................................20 
5.5.1.1. 
Primary Analysis for Primary Efficacy Variable ........................................................20 
5.5.1.2. 
Sensitivity Analyses for Primary Efficacy Variable ...................................................21 
5.5.2. 
Analysis of Secondary Efficacy Variables .................................................................21 
5.5.3. 
Analysis of Additional Efficacy Variables .................................................................21 
5.5.4. 
Control of Multiplicity ................................................................................................22 
5.5.5. 
Subgroup Analyses .....................................................................................................23 
5.6. 
Analysis of Safety Data ..............................................................................................23 
5.6.1. 
Adverse Events ...........................................................................................................23 
5.6.2. 
Surgeries .....................................................................................................................24 
5.6.3. 
Clinical Laboratory .....................................................................................................24 
5.6.4. 
Vital Signs ..................................................................................................................24 
5.6.5. 
Ocular Safety Measures ..............................................................................................24 
5.7. 
Analysis of Drug Concentration Data .........................................................................24 
5.8. 
Pharmacokinetics and Pharmacokinetics/Pharmacodynamics Analyses ....................24 
5.9. 
Analysis of Immunogenicity Data ..............................................................................25 
5.9.1. 
Analysis of ADA Data ................................................................................................25 
6. 
DATA CONVENTIONS ............................................................................................25 
6.1. 
Definition of Baseline .................................................................................................25 
6.2. 
Unscheduled Assessments ..........................................................................................25 
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6.3. 
Handling of Patients who Discontinue .......................................................................26 
6.4. 
Handling of Missing Data ...........................................................................................26 
6.4.1. 
General Rules ..............................................................................................................26 
7. 
INTERIM ANALYSIS ...............................................................................................26 
8. 
SOFTWARE ...............................................................................................................27 
9. 
REFERENCES ...........................................................................................................28 
10. 
APPENDIX.................................................................................................................29 
10.1. 
Schedule of Time and Events: ....................................................................................29 
10.2. 
Summary of Statistical Efficacy Analyses .................................................................33 
10.3. 
Calculation of confidence intervals using Mantel-Haenszel weighting 
scheme ........................................................................................................................34 
 
LIST OF FIGURES 
Figure 1: 
 Study flow diagram ....................................................................................................13 
 
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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
AE 
Adverse Event 
ALT  
Alanine Aminotransferase 
AST  
Aspartate Aminotransferase 
ATC 
Anatomical Therapeutically Chemical 
BUN 
Blood Urea Nitrogen 
CMH 
Cochran-Mantel-Haenszel 
CRF 
Case Report Form 
DMC 
Data Monitoring Committee  
DWFI 
Digital Wide-Field Images 
EOS 
End of Study 
FA 
Fluorescein Angiography 
FAS 
Full Analysis Set 
FP  
Fundus Photography 
ICF 
Informed Consent Form 
ICH 
International Conference of Harmonization 
IOP 
Intraocular Pressure 
IVT 
Intravitreal 
LDH 
Lactate Dehydrogenase 
LOCF 
Last Observation Carried Forward 
MedDRA 
Medical Dictionary for Regulatory Activities 
(MedDRA) HLT 
High Level Term 
(MedDRA) LLT 
Low Level Term 
(MedDRA) PT 
Preferred Term 
(MedDRA) SOC 
System Organ Class 
PPS 
Per Protocol Set 
RBC 
Red Blood Cell 
RC 
Reading Center 
ROP 
Retinopathy of Prematurity 
SAE 
Serious Adverse Event 
SAF  
Safety Analysis set 
SAP 
Statistical Analysis Plan 
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TEAE 
Treatment-emergent Adverse Event 
UPCR 
Urine Protein Creatinine Ratio 
VEGF 
Vascular Endothelial Growth Factor 
VEGFR1 
Vascular Endothelial Growth Factor Receptor 1 
VEGFR2 
Vascular Endothelial Growth Factor Receptor 2 
VEGF-A 
Vascular Endothelial Growth Factor-A  
WBC 
White Blood Cell 
WHO 
World Health Organization 
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DOCUMENT VERSION HISTORY 
Version  
Date 
Version History 
1.0 
02OCT2019 
Initial Version 
2.0 
21FEB2020 
Purpose: Update to incorporate the following revisions 
in response to feedback from the Food and Drug 
Administration (FDA): 
Section 3.4: Add the population of All Randomized Set  
Section 5.5.1.1: Define that patients who discontinue 
due to an adverse event between Week 40 and Week 52 
will be considered non-responders. 
Section 5.5.1.2: Add a sensitivity analysis for the 
primary variable to be performed based on all 
randomized patients.  
3.0 
13DEC2020 
Purpose: Update to incorporate the following revisions 
in response to feedback from the Food and Drug 
Administration (FDA): 
Section 2.1 Add the power for one-sided 0.025 
significance level. 
Section 5.5.1.1 Add one-sided 97.5% confidence 
intervals as supportive analysis. 
4.0 
05MAY2021 
Purpose: Update to incorporate the following revisions 
in response to feedback from FDA: 
Section 1.2.4. Document the changes from the 
statistical section in the protocol  
Section 5.1. Change the significant level to 2.45% (one-
sided test) (reflecting an alpha adjustment of 0.001 
(two-sided) for the DMC assessments) 
Section 5.5.1.1. Change confidence interval from one-
sided 95% to two-sided 95.1% and remove supportive 
analysis of one-sided 97.5% confidence interval.  
Section 5.5.4. Clarify the testing hierarchy for 
superiority test of primary endpoint  
Section 7. Add the alpha adjustment for Data 
Monitoring Committee (DMC) safety assessments. 
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1. 
OVERVIEW 
The purpose of the statistical analysis plan (SAP) is to ensure the credibility of the study results 
by pre-specifying the statistical approaches for the analysis of the study.  The statistical 
evaluation will be done according to the specifications given in the protocol and, if applicable, 
the corresponding amendments.  This SAP covers analyses through end of study visit (52-weeks 
of chronological age) only. 
1.1. 
Background/Rationale 
As stated in the study protocol, Retinopathy of prematurity (ROP) is a proliferative vascular 
retinopathy caused by an abnormal development of the vascularization of the peripheral retina in 
premature infants. It mainly affects newborns with a preterm gestational age (≤32 weeks) and 
very low birth weight (≤1500 g). Approximately 10% to 11% of all newborn infants are born 
preterm globally. Among premature infants, the incidence of ROP ranges from 20% to 30%, 
increasing for younger gestational ages. The prevalence of blindness caused by ROP in 
developed nations ranges between 6% and 18%, while in developing nations the estimate is 
>20%. Epidemiological studies have also identified increased risks for ROP due to genetic 
variants and environmental factors, such as oxygen exposure. 
Vascular endothelial growth factor (VEGF) is up-regulated under ischemic conditions. As ROP 
is characterized by incomplete vascularization of the retina in premature infants, it has also been 
associated with increased levels of VEGF. Vascular endothelial growth factor-A (VEGF-A) is a 
member of the VEGF family of angiogenic factors that acts through activation of the receptor 
tyrosine kinases vascular endothelial growth factor receptor 1 (VEGFR1) and vascular 
endothelial growth factor receptor 2 (VEGFR2) to increase mitosis, chemotaxis, and vascular 
permeability in endothelial cells. Upregulation of VEGF-A due to ischemia in the avascular 
retina may induce pathologic neovascularization and subsequently lead to retinal detachment and 
blindness, as seen in late-stage patients. Aflibercept inhibits these effects of VEGF by acting as a 
soluble decoy receptor that binds VEGF-A with higher affinity than the natural receptors and 
thereby inhibits the binding and activation of these cognate VEGF receptors. Aflibercept also 
inhibits the effects of placental growth factor, which binds only to VEGFR1. Placental growth 
factor can synergize with VEGF-A, enhancing these processes of pathologic neovascularization, 
and is also known to promote leukocyte infiltration and vascular inflammation. Based on the its 
mode of action, aflibercept has a high potential to become an effective treatment option for the 
treatment of ROP, and several reports of off-label clinical use have shown positive efficacy 
outcomes without indicating new safety concerns. 
This Phase 3, multicenter, randomized, 2-arm open-label study will assess the efficacy, safety, 
and tolerability of intravitreal (IVT) aflibercept compared to laser in patients (premature infants) 
diagnosed with ROP. 
1.2. 
Study Objectives 
1.2.1. 
Primary Objectives 
The primary objective of the study is to assess the efficacy of aflibercept compared to laser in 
patients diagnosed with ROP. 
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1.2.2. 
Secondary Objectives 
The secondary objectives of the study are: 
• To assess the need for a second treatment modality 
• To assess the recurrence of ROP in the study 
• To assess the safety and tolerability of aflibercept 
1.2.3. 
Exploratory Objectives 
The exploratory objectives of the study are: 
• To further characterize the anatomical effects of treatment with aflibercept in patients 
with ROP 
• To further investigate the study intervention (eg, mode-of-action-related effects 
and/or safety) relevant to this disease process 
• To assess the treatment burden of aflibercept and laser 
• To characterize the concentrations of free and bound aflibercept in plasma over time 
• To describe the potential immunogenicity of aflibercept 
1.2.4. 
Modifications from the Statistical Section in the Final Protocol 
To incorporate the feedback from FDA, the followings are modified from the statistical section 
in the final protocol.  
For the analysis of primary efficacy variable, the confidence interval is updated from one-sided 
95% to two-sided 95.1%, An alpha adjustment of 0.001 is made to the overall alpha at the final 
analysis in order to account for the DMC safety assessments. 
1.2.5. 
Modifications from the Approved Statistical Analysis Plan 
Not applicable 
2. 
INVESTIGATION PLAN 
2.1. 
Sample Size and Power Considerations 
The RAINBOW [1] study compared Ranibizumab 0.1 mg and 0.2mg to Laser, in the treatment 
of ROP. The primary endpoint is the absence of both active ROP and unfavorable structural 
outcomes at 24 Weeks. From the RAINBOW study, the response rate was 66.1% for the laser 
group and 88.1% for the 0.2 mg ranibizumab group in zone II disease. Furthermore, based on the 
clinical evidence for the aflibercept investigator-initiated studies (Salman, 2015[2]) (Huang, 
2018[3]) (Sukgen, 2018[4]) (Sidorenko, publication in progress), the response rates range all the 
way up to 100% (with at least 2 studies demonstrating a 100% response rate in terms of 
favorable anatomic structural outcomes/prevention of unfavorable anatomic structural outcomes) 
with intravitreal aflibercept doses ranging from 0.4 mg to 1 mg. Given the above data, an 
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estimated response rate for the primary efficacy variable of 90% for the aflibercept group and 
66.1% for the laser group is considered to be a reasonable assumption.  
A sample size of 84 patients in the aflibercept group and 28 patients in the laser group 
(randomized in a 3:1 ratio) will provide 92% power for testing non-inferiority (NI) of aflibercept 
versus laser at a one-sided 0.05 significance level assuming non-inferiority margin of 5% (See 
Section 5.1 for statement of the statistical hypotheses and justification of the NI margin). In 
addition, this sample size (a total of 112 patients randomized in a 3:1 ratio to aflibercept injection 
or laser, respectively) will provide 86% power for one-sided 0.025 significance level.  
The sample size calculation was computed using the binomial non-inferiority two sample test 
from the commercial software EAST version 6.4.1. 
2.2. 
Study Design and Randomization 
This is a phase 3, multicenter, randomized, 2-arm, open-label clinical study to assess the 
efficacy, safety, and tolerability of intravitreal (IVT) aflibercept versus laser in patients with 
ROP. The study consists of screening/baseline (1 or 2 visits), a treatment period (including 
potential retreatment and rescue treatment), and a final visit at week 52 of chronological age 
(Figure 1). 
The study also includes an optional fluorescein angiography (FA) sub-study, which includes 
optional FA at baseline, and mandatory FA at week 52 of chronological age. 
Successful screening requires the presence of treatment-naïve ROP classified by the investigator 
according to the International Classification for Retinopathy of Prematurity in at least 1 eye with 
1 of the following retinal findings: 
• Zone I Stage 1 plus, 2 plus, 3 non-plus or 3 plus, or 
• Zone II Stage 2 plus or 3 plus, or 
• AP-ROP 
One or both eyes can be treated according to the investigator’s assessment of the study’s 
eligibility criteria. If both eyes are eligible, they will be assigned to the same treatment group. 
The patient who starts the study with only 1 eligible eye should be kept under observation 
according to the local ROP screening guidelines or at every study visit, whichever is more 
frequent. Second eyes that develop ROP requiring treatment during the study should receive 
treatment according to the randomization assignment of the first eye. If the second eye is treated 
within 8 weeks of the initial treatment of the first eye, the second eye will be included in the 
efficacy analysis. 
For the primary efficacy endpoint, the patient will be the experimental unit. If both eyes are 
eligible and treated within 8 weeks, the success of the primary efficacy endpoint will be 
determined by both eyes. If both eyes are eligible but the second eye is treated outside 8 weeks 
of the initial treatment of the first eye, or only one eye is eligible, then the success of the primary 
endpoint will be determined by one eye. 
Color fundus photography (FP) with digital wide-field images (DWFI) of the retina will be taken 
before any treatment is applied. These images will be submitted to a central reading center (RC) 
as soon as possible for confirmation of ROP staging, and if deemed eligible, treatment should be 
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administered. However, due to the urgency of treatment for this medical condition, in situations 
where the investigator considers that awaiting a response from the RC and thus delaying 
treatment may be detrimental to the outcome, treatment is allowed to be administered 
immediately after the images are acquired, before availability of the RC confirmation of ROP 
staging. In this case, the investigator's assessment of the staging will be used in stratification. 
Patients will be randomized 3:1 to treatment with either aflibercept injection or laser, 
respectively, stratified by ROP classification in Zone I, Zone II, or AP-ROP according to 
investigator assessment. 
Aflibercept group: 
Patients randomized to aflibercept will receive a single IVT injection of aflibercept 
0.4 mg/0.01 mL per eligible eye at baseline. 
Thereafter, if required, up to 2 additional IVT injections of aflibercept 0.4 mg/0.01 mL may be 
administered in each eye if the following retreatment criteria are met: 
• Presence of ROP requiring treatment AND 
• The interval since the last aflibercept IVT injection is ≥28 days 
Rescue treatment with laser may be performed if 1 of the following conditions is met: 
• Worsening of ROP compared to the examination immediately preceding the previous 
aflibercept injection, during the 27 days following the aflibercept injection 
• Presence of ROP requiring treatment after the patient already received a total of 3 
aflibercept injections 
Laser Group: 
Patients randomized to laser will undergo treatment in each eligible eye at baseline. Laser 
ablation should be as complete as possible as judged by the investigator. In case multiple 
sessions are necessary within 1 week from baseline, they will be counted as a single treatment. 
Treatment will be applied to the entire avascular peripheral retina. Treatment should be kept well 
away from the fovea. 
Supplementary laser treatments are allowed during the study. Retreatment with laser is allowed if 
both of the following criteria are met: 
• Presence of ROP requiring treatment 
• Fundus examination reveals that laser treatment is incomplete as judged by the 
investigator 
Rescue treatment with aflibercept 0.4 mg/0.01 mL is allowed if the fundus examination reveals 
that laser treatment is complete as judged by the investigator and if 1 of the following conditions 
is met: 
• Worsening of ROP compared to the most recent pre-laser examination 
• Persistence of ROP requiring treatment 
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Patients who initiate aflibercept rescue treatment may receive additional aflibercept injections 
according to the aflibercept group treatment regimen. 
The study event table is presented in Appendix 10.1. 
Figure 1: 
 Study flow diagram 
 
3. 
ANALYSIS POPULATIONS 
In accordance with guidance from the International Conference of Harmonization of Technical 
Requirements for Registration of Pharmaceuticals for Human Use (ICH) guideline 
ICH E9 Statistical Principles for Clinical Trials (ICH,1998 [5]), the following analysis 
populations will be used for all statistical analysis: 
3.1. 
Full Analysis Set 
The full analysis set (FAS) will include all randomized patients who received any study 
treatment. The analysis on the FAS will be performed according to the treatment assigned at 
baseline (as randomized).  
3.2. 
Safety Analysis Set 
The safety analysis set (SAF) includes all randomized patients who received any study 
treatment (active or laser); it is based on the treatment actually received (as treated). Treatment 
compliance/administration and all clinical safety variables will be analyzed using the SAF. 
3.3. 
Per Protocol Set 
The Per Protocol Set (PPS) includes all patients in the FAS who had no important protocol 
deviations. Analysis of the PPS will be performed according to the treatment actually received 
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(as treated). The PPS will be used for sensitivity analysis of primary efficacy endpoint. The 
protocol deviation plan will be finalized prior to start of study enrollment.  
3.4. 
All Randomized Set 
All Randomized Set will include all randomized patients regardless receiving study treatment or 
not. The analysis on the all randomized patients will be performed according to the treatment 
assigned at baseline (as randomized). 
3.5. 
Pharmacokinetic Analysis Set 
The Pharmacokinetic (PK) Analysis Set includes all patients who received any study treatment 
and who had a at least 1 non-missing result following the first dose of study treatment. 
3.6. 
Immunogenicity Analysis Sets 
The ADA analysis set (AAS) includes all patients who received any study treatment and had at 
least one non-missing ADA result from the Aflibercept ADA assay after first dose of the study 
treatment.  Patients will be analyzed according to the treatment actually received. 
4. 
ANALYSIS VARIABLES 
4.1. 
Demographic and Baseline Characteristics 
Demographic and baseline assessments to be summarized will include: 
• Chronological age at randomization (date of randomization - date of birth + 1) 
• Gestational age at birth (in weeks and days) 
• Post-menstrual age at randomization (Gestational age at birth + Chronological age at 
randomization) 
• Gender 
• Race 
• Body weight, head circumference and body length at birth and at baseline 
• Vital Signs (Baseline pulse rate, systolic blood pressure, diastolic blood pressure and 
temperature) 
• Baseline Intraocular pressure (IOP) 
• ROP classification (Zone I, Zone II, plus status and AP-ROP) 
• O2 supplementation at baseline (yes, no) 
• History of sepsis (yes, no) 
• History of necrotizing enterocolitis (yes, no) 
• History of intraventricular hemorrhage (yes, no) 
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• Number of patients with two/one eye(s) enrolled 
4.2. 
Medical History 
Maternal and patient Medical history will be coded according to latest available version of 
Medical Dictionary for Regulatory Activities (MedDRA). 
4.3. 
Pre-Treatment / Concomitant Medication 
Medications taken during the study will be recorded and will be coded to Anatomical 
Therapeutically Chemical (ATC) codes according to the World Health Organization Drug 
Dictionary (WHO Drug Dictionary) B3 Global 201903 enhanced version provided by Bayer 
Health Care. 
Medications will be summarized as follows: 
• Maternal prior medications taken during pregnancy (i.e. medication taken by the 
mother with start day prior to date of birth of child but after conception) 
• Prior medication taken by the breastfeeding mother (i.e. medication taken by the 
mother with start date after date of birth but prior to the start of study treatment and 
end date prior to the start of study treatment)  
• Prior medication taken by the patient   
• Concomitant medication taken by the breastfeeding mother  
• Concomitant medication taken by the patient 
The prior, concomitant and new medication will be summarized by ATC class (ATC level 1) and 
subclass (ATC level 2). 
Variables for concomitant medication description and analysis will include Generic name, ATC 
level codes, Indication, Dose/Dose Unit, Frequency, Route, start/end date and study day, 
Duration, Ongoing. 
4.4. 
Exposure and Additional Treatment to Study Treatment 
Exposure 
For each patient, the following variables will be used to examine exposure to study treatment 
(rescue treatment will not be taken into account here): 
• Total number of treatment (aflibercept or laser) by treatment group 
• For patients who receive rescue treatment, frequency of treatment will be summarized 
• Time required to perform treatment 
• Requirement for sedation or general anesthesia  
• Injection volume  
• Number of burns, wave length, spot sizes, power and duration of laser pulse (for laser 
treatment) 
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4.5. 
Efficacy Variable 
4.5.1. 
Primary Efficacy Variable (s) 
The primary efficacy variable is the proportion of patients with the absence of both active ROP 
and of unfavorable structural outcomes at 52 weeks of chronological age, as determined by the 
Investigator. For patients with both eyes enrolled in the study, both eyes must meet the endpoint. 
Active ROP is defined as ROP requiring treatment (according to the inclusion criteria). 
Unfavorable structural outcomes are defined as retinal detachment, macular dragging, macular 
fold, or retrolental opacity. 
Furthermore, patients will be considered as non-responders if rescue treatment is given in the 
study eye(s).   
4.5.2. 
Secondary Efficacy Variable(s) 
The secondary efficacy variables are: 
• Proportion of patients requiring intervention with a second treatment modality from 
baseline to week 52 of chronological age 
• Proportion of patients with recurrence of ROP through week 52 of chronological age.  
Recurrence of disease is defined further in Section 5.5.2. 
4.5.3. 
Exploratory Efficacy Variable(s) 
The exploratory efficacy variables are: 
• Time to recurrence of ROP 
• Time to intervention with a second treatment modality for ROP or to development of 
unfavorable structural outcomes 
• Proportion of patients with completion of vascularization of the peripheral retina to 
within 1 disc diameter of the ora serrata on FP at week 52 of chronological age 
• Presence of retinal vascularization and leakage on fluorescein angiography (FA) at 
week 52 of chronological age in patients participating in the FA substudy 
• Time to completion of retinal vascularization 
• Proportion of patients with regression of plus disease, regression of pre-retinal 
vascularized ridge, and progression of retinal vascularization beyond the ridge from 
baseline to week 52 of chronological age 
• Proportion of patients with progression to stage 4 or 5 ROP from baseline to week 52 
of chronological age 
• Evaluation of visual function at week 52 of chronological age 
• Time required to perform aflibercept injection or initial laser treatment 
• Proportion of patients with requirement for sedation or general anesthesia to complete 
laser or aflibercept injection 
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4.6. 
Safety Variables 
4.6.1. 
Adverse Events and Serious Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a patient administered a study 
treatment which may or may not have a causal relationship with the study treatment. 
Adverse events will be collected at each visit from the time of informed consent signature until 
the end of the study.  If the patient withdraws from the study during the screening, AEs will be 
collected up until the patient withdraws.  If the patient withdraws at any point after receiving the 
first dose of study treatment, AEs will be collected up until 30 days after the last dose of study 
treatment or the termination visit, whichever is later. 
The focus of AE reporting in the clinical study report will be on Treatment-Emergent Adverse 
Events (TEAEs) :  TEAEs are defined as AEs that developed or worsened during the on-
treatment period, that is, AEs that are observed or reported after first and not later than 30 days 
after last administration of study treatment (laser or aflibercept injection) in the study eye(s), 
laser or aflibercept rescue treatment in the fellow eye.   
Other variables for AE description and analysis will include AE Verbatim Term, AE start date 
and end date/ongoing and corresponding study day, AE Duration, relationship of AE to study 
treatment, relationship of AE to study conduct, seriousness, intensity, action due to AE, 
treatment of AE and outcome. 
4.6.2. 
Surgeries 
All the surgeries after informed consent are collected on the CRF and are coded by MedDRA. 
The following variables will be tabulated by MedDRA preferred term: 
• Treatment emergent surgery is defined as surgery performed on or after the start of 
study treatment  
− Ocular treatment emergent surgery for study eye(s) and fellow eye 
− Non-ocular treatment emergent surgery 
4.6.3. 
Laboratory Safety Variables 
Clinical laboratory variables will include the following: 
• Blood chemistry panel:  Sodium, Total protein, serum, Total bilirubin, Potassium, 
Creatinine, Total cholesterol, Chloride, Blood urea nitrogen (BUN), Triglycerides, 
Carbon dioxide, Aspartate aminotransferase (AST), Uric acid, Calcium, Alanine 
aminotransferase (ALT), Creatine phosphokinase (CPK), Glucose, Alkaline 
phosphatase, Albumin, Lactate dehydrogenase (LDH) 
• Hematology panel:  Hemoglobin, Hematocrit, Red blood cells (RBC), White blood 
cells (WBC), Red Cell Indices, Platelet count, Differential:  Neutrophils, 
Lymphocytes, Monocytes, Basophils, Eosinophils 
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• Urinalysis:  Color, Glucose, RBC, Clarity, Blood, Hyaline and other casts, pH, 
Bilirubin, Bacteria, Specific gravity, Leukocyte esterase, Epithelial cells, Ketones, 
Nitrite, Crystals, Protein, WBC, Yeast 
4.6.4. 
Vital Signs 
Variables of analysis for vital signs include temperature, pulse, blood pressure, and respiration 
rate. 
4.6.5. 
Ocular Safety Measures 
Variables of analysis for ocular safety measures include: 
• Proportion of patient with increased intraocular ocular pressure (IOP) 
− ≥ 10 mmHg increase in IOP measurement from baseline to any pre-dose 
measurement 
− > 21 mmHg for any pre-dose measurement 
− ≥ 25 mmHg for any pre-dose measurement 
− ≥ 35 mmHg at any time 
Post dose IOP measurement should be the last IOP recorded. 
4.7. 
Pharmacokinetic Variables 
The PK variable is the concentration of free or bound aflibercept in plasma at each time point. 
4.8. 
Immunogenicity Variables 
The immunogenicity variables are ADA status, titer, and time-point/visit. Serum samples for 
ADA will be collected at the clinic visits specified in Appendix 10.1.   
5. 
STATISTICAL METHODS 
All efficacy and safety variables will be summarized descriptively with appropriate statistics:  
categorical variables by frequency (absolute and relative frequencies) and continuous variables 
by sample statistics (i.e. mean, standard deviation, minimum, median, quartiles and maximum).  
Continuous variables will be described by visit and as change from Baseline, if applicable. 
5.1. 
Statistical Hypothesis 
This study will examine the following hypothesis for the primary efficacy variable regarding the 
proportion of patients with both the absence of active ROP and unfavorable structural outcomes 
at 52 weeks of chronological age. Statistical testing will be conducted to demonstrate the non-
inferiority of the aflibercept group to the laser group, with a non-inferiority margin of 5% and a 
significance level of 2.45% (one-sided test) (reflecting an alpha adjustment of 0.001 (two-sided) 
for the DMC assessments). 
H0: pt ≤ pc - 5% versus H1: pt ≥ pc - 5% 
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where pt is the true proportion of patients with absence of active ROP and unfavorable structural 
outcomes at week 52 of chronological age for the aflibercept treated group, and pc is the true 
proportion of patients with absence of active ROP and unfavorable structural outcomes at week 
52 of chronological age for the laser control group. 
In this trial, laser is the active control arm.  From the RAINBOW study, the success rate for laser 
was 66.2% (95% CI: 55.0% to 77.4%), while the success rate for a putative placebo (which has 
little to no efficacy) is assumed to be near 0%.  The 95-95 approach described in Rothman 
[6,7,8] was used to determine the non-inferiority (NI) margin for the study.  Using this method, 
the margin, M1 (also known as statistical margin), is conservatively assumed to be the lower 
limit of 95% CI for the treatment difference (laser – putative placebo), i.e., 55%.  The smaller 
margin, M2=5% (also known as the clinical margin) was prespecified such that a large fraction 
of the active control (laser) treatment effect is preserved for treating ROP.  The proposed NI 
margin of 5% is the smaller of M1 and M2, and preserves at least 90.9% of the control treatment 
effect. Therefore, this NI margin is adequate and justified.  
5.2. 
Subject Disposition 
The following categories for patient disposition will be summarized descriptively: 
• The total number of screened patients:  met the inclusion criteria regarding the target 
indication and signed the informed consent form (ICF) 
• The total number and percentage of randomized patients:  received a randomization 
number 
• The number and percentage of patients in each analysis set 
• The total number and percentage of patients who discontinued the study with the 
reasons for discontinuation 
The following listings will be provided to assess the patient disposition: 
• A listing of patients treated but not randomized and patients randomized but not 
treated if any 
• A listing of patients who received rescue treatment in the study eye(s) 
• A listing of patients who were withdrawn from the study, along with reasons for 
discontinuation 
• Listing of important protocol deviations:  violation of inclusion/exclusion criteria; 
post-enrollment deviations which will impact assessment of efficacy endpoints 
5.3. 
Demographics, Baseline Characteristics, Medical History, and Prior/ 
Concomitant Medication 
Demographic data and baseline characteristics variables described in Section 4.1 will be 
summarized using descriptive statistics for SAF and FAS. 
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Medical history is evaluated for SAF by a frequency table, showing number of patients with 
medical history findings by primary system organ class (SOC), high level term (HLT) by 
MedDRA terms. 
Prior/concomitant medication will be summarized by WhoDrug B3 Global 201903ATC codes 
(ATC 3-digit class and ATC 5-digit subclass) for medication taken during the study.  Separate 
frequency tables will be displayed for patients with prior medications, new medications and 
concomitant medications by the time periods described in Section 4.3. 
5.4. 
Extent of Study Treatment Exposure  
The variables for dose exposure described in Section 4.4 will be summarized in the SAF and 
FAS populations, using descriptive statistics. 
5.5. 
Analyses of Efficacy Variables 
Efficacy analyses of all efficacy variables defined in Section 4.5 will be conducted using the 
FAS population. PPS population will be used as sensitivity analysis for primary efficacy 
variable. 
5.5.1. 
Analysis of Primary Efficacy Variable(s) 
5.5.1.1. 
Primary Analysis for Primary Efficacy Variable 
The primary efficacy variable analysis will be conducted on the FAS. The primary analysis is a 
statistical evaluation of non-inferiority of aflibercept vs. laser at week 52 of chronological age, 
with respect to the primary efficacy variable (i.e., absence of both active ROP and of unfavorable 
structural outcomes; see Section 4.5.1). The non-inferiority margin is set at 5%. Superiority 
testing will follow if non-inferiority is established. 
If the patient data are not available at end of study (EOS) visit, then data available from the week 
40 of chronological age visit will be carried forward (LOCF) to the EOS visit for analysis except 
the following two cases:  
• If the patient discontinues at or before the week 40 visit, no data will be carried 
forward and the patient will be considered a non-responder.  
• If patients discontinue the study due to AEs between week 40 and week 52, then these 
patients will be considered as non-responders.  
For patients with both eyes enrolled in the study, both eyes must meet the endpoint. Patients with 
only one study eye enrolled will be responders if the respective eye responds. 
The statistical analysis will be performed using the Cochran-Mantel-Haenszel method stratified 
by baseline ROP status. The two-sided 95.1% Mantel-Haenszel confidence intervals (reflecting 
an alpha adjustment of 0.001 for the DMC assessments) using normal approximation of the 
difference of response rates between the aflibercept group and the laser group will be calculated. 
Aflibercept will be considered to be non-inferior to laser if the lower confidence bound of the 
difference lies above -5%.  Furthermore, if the lower confidence bound lies above 0%, 
superiority of Aflibercept may be declared.  
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The primary analysis for this endpoint will be based on the investigator assessment.  
The components of the primary efficacy variable, i.e., number and percentage of patients with 
absence of active ROP and/or number and percentage of patients with unfavorable outcomes will 
also be presented. 
5.5.1.2. 
Sensitivity Analyses for Primary Efficacy Variable 
The primary efficacy variable will be analyzed using the PPS population and all randomized 
population as sensitivity analyses. 
Observed case (OC) analysis will be conducted as supplementary analysis. Only observed values 
will be used for analysis. Patients will be considered as non-responders if rescue treatment is 
given. 
The same analysis for the primary endpoint will be performed based on the central RC data as a 
sensitivity analysis.  
5.5.2. 
Analysis of Secondary Efficacy Variables 
The following secondary efficacy variables will be analyzed using the same method as for the 
analysis of the primary efficacy variable. Patients will be counted as “event” if at least one eye 
satisfies the criteria. 
• Proportion of patients requiring intervention with a second treatment modality from 
baseline to week 52 of chronological age 
• Proportion of patients with recurrence of ROP through week 52 of chronological age 
Recurrence of disease is defined as the reappearance of the disease requiring further treatment 
(including retreatment or rescue), where both “presence of ROP” and “presence of active ROP 
requiring treatment” are marked as “Yes”, after initial regression. Here, the initial regression is 
defined as, at particular visit, absence of ROP or ROP treatment not required for active ROP, i.e., 
presence of ROP is marked as “No” or the presence of active ROP requiring treatment is marked 
as “No”. 
5.5.3. 
Analysis of Additional Efficacy Variables 
The following additional efficacy variables will be summarized descriptively by frequency 
tables.  
• Proportion of patients with completion of vascularization of the peripheral retina to 
within 1 disc diameter of the ora serrata on FP at week 52 of chronological age 
− It will be summarized per eye and per patient. 
• Presence of retinal vascularization and leakage on fluorescein angiography (FA) at 
week 52 of chronological age in patients participating in the FA substudy 
− It will be summarized per eye. 
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• Proportion of patients with regression of plus disease, regression of pre-retinal 
vascularized ridge, and progression of retinal vascularization beyond the ridge from 
baseline to week 52 of chronological age  
− It will be summarized per eye. 
• Proportion of patients with progression to stage 4 or 5 ROP from baseline to week 52 
of chronological age 
− It will be summarized per eye and per patient. 
• Evaluation of visual function at week 52 of chronological age  
− It will be summarized per eye and per patient. 
• Proportion of patients with requirement for sedation or general anesthesia to complete 
laser or aflibercept injection  
− It will be summarized per patient. The number of episode(s) will also be 
summarized. 
Visual function will be presented by descriptive statistics for the following variables: 
• fixation (central, steady and maintained) – Yes/No 
• fixing and following a 5-cm toy – Yes/No 
• in case visual function cannot be assessed with fixation, visual evoked potentials can 
be used for instance to evaluate if the visual function is normal or abnormal – 
Normal/Abnormal 
• cycloplegic refraction (sphere, cylinder, axis, pseudophakia or intraocular vision 
correction) - Spherical equivalent 
• ocular motility tests (if motility test done: assessment of ocular palsy with affected 
nerves, assessment of strabismus, and nystagmus). – Normal/Abnormal 
The following time to event variables will be analyzed by patient level using the Kaplan-Meier 
estimates and displayed descriptively by treatment group. For patients with both eyes enrolled in 
the study, the time to event is defined as the time from randomization to the first event occurred 
in either eye. 
• Time to recurrence of ROP 
• Time to intervention with a second treatment modality for ROP or to development of 
unfavorable structural outcomes 
• Time to completion of retinal vascularization 
• Time required to perform aflibercept injection or initial laser treatment 
5.5.4. 
Control of Multiplicity 
To control the overall Type I error at α=0.05 (two-sided), an adjustment of 0.001 will be made 
for the DMC safety assessments.  Therefore, at the final analysis, all nominal p-values will be 
compared to α = 0.049 and two-sided 95.1% confidence intervals will be reported. 
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If non-inferiority of the primary endpoint is declared significant, a hierarchical procedure for 
testing superiority will be used for the analysis of the secondary endpoints to control the overall 
alpha error rate at the 0.05 level based on the following order.  
• Proportion of patients requiring intervention with a second treatment modality from 
baseline to week 52 of chronological age 
• Proportion of patients with recurrence of ROP through week 52 of chronological age 
The superiority of the primary endpoint will be tested, after testing for superiority of both 
secondary efficacy endpoints. 
5.5.5. 
Subgroup Analyses 
Subgroup analyses will be performed on the FAS population using descriptive statistics for 
primary efficacy based on the ROP status subgroup. 
5.6. 
Analysis of Safety Data 
The safety variables as described in Section 4.6 will be analyzed on SAF population. An 
additional pooled analysis may be performed between this study and studies #20090 
(FIREFLEYE) and #20275 (FIREFLEYE NEXT) being conducted outside of the US by 
Regeneron’s development partner, Bayer. 
5.6.1. 
Adverse Events  
TEAE summaries will be constructed displaying frequencies and proportions of patients 
reporting TEAEs within each SOC in decreasing order of total frequency according to the 
numbers of patients reporting the SOC and the preferred term (PT) within the SOC (not number 
of reports). The following categories will be summarized: 
• Ocular TEAEs in the study eye(s)  
• Ocular TEAEs in the fellow eye 
• Non-ocular TEAEs  
For the ocular TEAEs in the study eye(s), the frequency of the events and the number of study 
eyes(s) will be displayed as well. For the ocular TEAEs in the fellow eye, the number of patients 
with one study eye enrolled in each treatment group will be used as denominator. 
Ocular TEAEs in the study eye(s) and non-ocular TEAEs will be further summarized for 
bilaterally and unilaterally treated patients separately. 
Serious TEAEs, treatment-related TEAEs, treatment-related Serious TEAEs, and TEAEs leading 
to discontinuation will be summarized in the same way as described for TEAE. 
TEAEs in the study eye related to the study conduct and those related to the study treatment will 
be summarized separately. 
An overall summary of the AE profile for aflibercept in this study will be provided.  A listing 
will be constructed that includes the patient identification, the treatment group, category of 
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TEAE (ocular study eye(s) or fellow eye, non-ocular), AE, MedDRA term, seriousness, severity, 
causality, elapsed time to onset, duration, and outcome. 
Beyond TEAEs, All AEs from first dose of study treatment to end of study will be summarized 
in the same way as described for TEAE. 
Subgroup analyses in TEAEs will be performed for the ROP status subgroup, for each of the 
following types of TEAE: 
Summaries (by SOC and PT) of patients with: 
• Ocular TEAEs study eye(s)  
• Non-ocular TEAEs 
• Serious ocular TEAEs study eye(s)  
• Serious non-ocular TEAEs 
5.6.2. 
Surgeries 
An overall summary of number of patients undergoing surgery as described in Section 4.6.2 will 
be given by treatment group. 
5.6.3. 
Clinical Laboratory 
Clinical laboratory data will be collected only at screening. Data listings will be provided for 
hematology, chemistry and urinalysis data. 
5.6.4. 
Vital Signs 
Baseline vital signs and change from Baseline for vital sign variables described in Section 4.6.4 
at each scheduled assessment visit will be summarized using descriptive statistics.  These will 
include the number of patients, mean, median, standard deviation, minimum, and maximum. 
5.6.5. 
Ocular Safety Measures 
Baseline IOP and change from Baseline in IOP to each scheduled assessment visit will be 
summarized with descriptive statistics for study eye(s) treated with Aflibercept.  Assessment of 
significant values or increases will be made and summarized for the proportion of patients with 
increased IOP in the study eye(s) with the categories defined in Section 4.6.5. 
5.7. 
 Analysis of Drug Concentration Data 
The plasma concentrations of free and bound aflibercept over time will be summarized by 
descriptive statistics. No formal statistical hypothesis testing will be performed. 
5.8. 
Pharmacokinetics and Pharmacokinetics/Pharmacodynamics 
Analyses 
Population PK and exposure-response analyses for blood pressure may be performed, as 
appropriate. 
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5.9. 
Analysis of Immunogenicity Data 
5.9.1. 
Analysis of ADA Data 
The immunogenicity variables described in Section 4.7 will be summarized using descriptive 
statistics. Listings of ADA positivity and titers presented by patient, time point, and study 
treatment received will be provided. Incidence of treatment-emergent and treatment-boosted 
ADA will be assessed as absolute occurrence (N) and percent of patients (%), grouped by study 
treatment received.  
Immunogenicity will be characterized by ADA responses and titers observed in patients in the 
ADA analysis set. ADA response categories and titer categories are defined as follows:  
− Pre-existing immunoreactivity, defined as either an ADA positive response in the 
aflibercept ADA assay at baseline with all post dose ADA results negative, OR a positive 
response at baseline with all post first dose ADA responses less than 4 -fold over baseline 
titer levels. 
− Treatment-emergent response, defined as a positive response in ADA assay post first 
dose when baseline results are negative or missing.  
− Treatment-boosted response, defined as a positive response in the aflibercept ADA assay 
post first dose that is greater than or equal to 4-fold over baseline titer levels, when 
baseline results are positive. 
Titer categories (Maximum titer values) 
− Low (titer <1,000) 
− Moderate (1,000 ≤ titer ≤ 10,000) 
− High (titer >10,000) 
6. 
DATA CONVENTIONS 
The following analysis conventions will be used in the statistical analysis. 
6.1. 
Definition of Baseline 
Unless otherwise specified, the Baseline assessment for all measurements will be the last 
available valid measurement taken prior to the administration of investigational product. 
6.2. 
Unscheduled Assessments 
Assessments taken outside of protocol allowable windows will be displayed according to the 
CRF assessment recorded by the investigator. 
Unscheduled assessments will not be included in the summaries. 
If more than one value is available for a given visit, the value for this visit actually used for 
statistical summaries and analyses is as follows: 
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• The last non-missing repeated measurement, if the respective visit is before the start 
of treatment 
• The first non-missing repeated measurement, if the respective visit is after the start of 
treatment 
If an early termination visit is performed within the window (±1 week) of the next scheduled 
visit after the last visit, the efficacy assessment will be re-slotted to the next scheduled visit. 
6.3. 
Handling of Patients who Discontinue 
Patients who discontinue this study will not be replaced.  The details for the handling of missing 
data due to patients who discontinue the study and study treatment are described in Section 6.4.1. 
6.4. 
Handling of Missing Data  
6.4.1. 
General Rules 
When appropriate, the following rules will be implemented so as not to exclude patients from 
statistical analyses due to missing or incomplete data: 
• Efficacy Variables 
For the primary, secondary and additional efficacy variables, missing observations will 
be imputed using LOCF if data are available from week 40 of chronological age visit. 
Otherwise, missing data will be considered as non-responders. The details are described 
in the efficacy analysis section (see Section 5.5). 
• AE variables  
For some AEs it is important to determine whether the AE started before or after the first 
active aflibercept injection.  If the AE start date is partially missing, it will be imputed by 
the latest possible date (considering other available data, e.g., stop date) to be 
conservative. 
• Prior/concomitant medication 
For the tabulation of prior and concomitant medication, partially missing start dates of the 
medication will be imputed by the earliest possible time point, partially missing stop 
dates will be imputed by the latest possible time point. 
7. 
INTERIM ANALYSIS 
No formal interim analysis is planned. An independent data monitoring committee (DMC) will 
review the safety to determine if the study shows unacceptable risks for the patients, and to 
discontinue the study if necessary. Detailed information regarding the DMC procedures will be 
explained in a separate DMC charter. 
An alpha adjustment of 0.001 will be made to the overall alpha at the final analysis in order to 
account for the DMC safety assessments. 
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8. 
SOFTWARE 
All analyses will be done using SAS Version 9.4 or higher. 
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9. 
REFERENCES 
1. 
RAINBOW Study: RAnibizumab Compared With Laser Therapy for the Treatment of 
INfants BOrn Prematurely With Retinopathy of Prematurity (RAINBOW).  
https://clinicaltrials.gov/ct2/show/NCT02375971 
2. 
Salman AG and Said AM. Structural, visual and refractive outcomes of intravitreal 
aflibercept injection in high-risk prethreshold type 1 retinopathy of prematurity. Ophth Res 
2015;53:15-20. 
3. 
Huang CY, Lien R, Wang NK, et al. Changes in systemic vascular endothelial growth 
factor levels after intravitreal injection of aflibercept in infants with retinopathy of 
prematurity. Graefes Arch Clin Exp Ophthalmol 2018;256, 479-487. 
4. 
Sukgen EA and Kocluk Y. Comparison of clinical outcomes of intravitreal ranibizumab 
and aflibercept treatment for retinopathy of prematurity. Graefe’s Archive for Clinical and 
Experimental Ophthalmology. Revised 2018 Apr 16. Available from: 
https://doi.org/10.1007/s00417-018-4168-5. 
5. 
ICH. (1998, February 5). ICH Harmonized tripartite guideline:  Statistical principles for 
clinical trials (E9).  International Conference on Harmonization of Technical Requirements 
for Registration of Pharmaceuticals for Human Use. 
6. 
Rothmann M, Li N, Chen G, et al: Design and analysis of non-inferiority mortality trials in 
oncology. Stat Med 2003, 22:239-264. 
7. 
Rothmann MD, Tsou HH: On non-inferiority analysis based on delta-method confidence 
intervals. J Biopharm Stat 2003, 13:565-583. 
8. 
United States Food and Drug Administration: Guidance for Industry Non-Inferiority 
Clinical Trials 2010. 
 
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10. 
APPENDIX 
10.1. 
Schedule of Time and Events: 
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10.2. 
Summary of Statistical Efficacy Analyses 
Endpoint 
Major Analysis, 
population 
Statistical Analysis 
Sensitivity 
Analysis 
Proportion of patients with absence of 
active ROP and of unfavorable 
structural outcomes at week 52 of 
chronological age  
LOCF, FAS, 
investigator 
assessment 
Non-inferiority of aflibercept vs. 
laser using CMH test adjusted by 
baseline ROP status 
OC, PPS, 
RC data 
Proportion of patients requiring 
intervention with a second treatment 
modality from baseline to week 52 of 
chronological age 
LOCF, FAS 
Superiority of aflibercept vs. laser 
using CMH test adjusted by 
baseline ROP status 
 
Proportion of patients with recurrence of 
ROP through week 52 of chronological 
age 
LOCF, FAS 
Superiority of aflibercept vs. laser 
using CMH test adjusted by 
baseline ROP status 
 
Time to recurrence of ROP 
FAS 
Kaplan-Meier estimate 
 
Time to intervention with a second 
treatment modality for ROP or to 
development of unfavorable structural 
outcomes 
 
FAS 
Kaplan-Meier estimate 
 
Completion of vascularization of the 
peripheral retina to within 1 disc 
diameter of the ora serrata on FP at 
week 52 of chronological age 
FAS 
Descriptive 
Statistics 
 
Assessment of retinal vascularization 
and leakage on fluorescein angiography 
(FA) at week 52 of chronological age in 
patients participating in the FA substudy 
FAS 
Descriptive 
Statistics 
 
Time to completion of retinal 
vascularization 
FAS 
Kaplan-Meier estimate 
 
Regression of plus disease, regression of 
pre-retinal vascularized ridge, and 
progression of retinal vascularization 
beyond the ridge from baseline to week 
52 of chronological age 
FAS 
Descriptive 
Statistics 
 
Progression to stage 4 or 5 ROP from 
baseline to week 52 of chronological 
age 
FAS 
Descriptive 
Statistics 
 
Evaluation of visual function at week 52 
of chronological age 
FAS 
Descriptive 
Statistics 
 
Time required to perform aflibercept 
injection or initial laser treatment 
FAS 
Kaplan-Meier estimate 
 
Requirement for sedation or general 
anesthesia to complete laser or 
aflibercept injection 
FAS 
Descriptive 
Statistics 
 
 
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10.3. 
Calculation of confidence intervals using Mantel-Haenszel weighting 
scheme 
The confidence intervals using the Mantel-Haenszel weighting scheme will be calculated 
according to the formulas given by Koch et al. (1990, p. 415 ff.)2, i.e. to compute confidence 
intervals for the difference in two binomial proportions obtained from a multicenter trial, we 
calculate a weighted difference and its associated variance using Mantel-Haenszel weighting 
scheme. 
For a multicenter study with h 2x2 tables, the weighted difference is: 
d = ( wh(phe - phs))/( wh ) 
where wh = nhenhs/(nhe+nhs) 
and 
phe = success rate for experimental treatment in stratum h  
 
phs = success rate for standard treatment in stratum h 
 
nhe = number of patients under experimental treatment in stratum h 
 
nhs = number of patients under standard treatment in stratum h 
The variance of the weighted difference is: 
var(d) =( wh2(phs(1-phs)/(nhs -1) + phe(1-phe)/(nhe – 1)))/( wh)2 
A large sample approximation is used to compute the confidence interval: 
 
CI = d ± z SQRT(var(d)) 
Where z is the α quantile of the standard normal distribution and SQRT is the square root 
function. 
                                        VV-RIM-00152939-1.0 Approved - 07 May 2021 GMT-5:00


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                                        VV-RIM-00152939-1.0 Approved - 07 May 2021 GMT-5:00