Conditions: COVID-19
Keywords: COVID-19 Plitidepsin
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 37
Healthy volunteers: no

Brief summary:
The primary objective of this study is to evaluate efficacy of plitidepsin in pre-specified groups of immunocompromised patients with symptomatic COVID-19 requiring hospital care versus control in terms of mortality.

Detailed description:

Arms:
arm 1: Best standard care (as per applicable local, institutional, national, supranational COVID-19 treatment guidelines) and plitidepsin (administered as a 60-minute intravenous (IV) infusion, every 24 hours for 3 consecutive days, at a dose of 2.5 mg) will be administered to participants of the following groups:

* Group 1 - Participants receiving immune-suppression due to haematopoietic or organ transplantation.
* Group 2 - Participants receiving B-cell depleting therapies.
* Group 3 - Participants receiving other immune-suppressive therapies.
* Group 4 - Other situations with immune deficiencies. arm 2: Best standard care (as per applicable local, institutional, national, supranational COVID-19 treatment guidelines) ± other regulatory-approved antiviral (if clinically indicated) will be administered to participants of the following groups:

* Group 1 - Participants receiving immune-suppression due to haematopoietic or organ transplantation.
* Group 2 - Participants receiving B-cell depleting therapies.
* Group 3 - Participants receiving other immune-suppressive therapies.

Interventions: intervention 1: Plitidepsin
Intervention descriptions: intervention 1: IV infusion over 60-minutes

Protocol document:
Prot_000.pdf: 
Protocol Number: AV-APL-B-002-22 
 
Official Title: A Multicentre, Open label, Randomised, Controlled, Basket, Pragmatic, Phase II, 
Clinical and Translational Study to Determine the Efficacy and Safety of Plitidepsin versus Control 
in Immunocompromised Adult Patients with Symptomatic COVID-19 requiring Hospital Care 
(NEREIDA) 
 
NCT Number: NCT05705167 
 
Document Date: 22 March 2023 


 
                                                                     
Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
                                                                          
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 1
 
 
 
 
SPONSOR 
Pharma Mar, S.A. 
Avda. de los Reyes, 1 
 
Polígono Industrial “La Mina”  
28770 Colmenar Viejo (Madrid) Spain 
Phone: + 34 91 846 6000  
Fax:       + 34 91 846 6003  
 
 
 
CLINICAL TRIAL PROTOCOL 
AV-APL-B-002-22 
A Multicentre, Open label, Randomised, Controlled, Basket, 
Pragmatic, Phase II, Clinical and Translational Study to Determine the 
Efficacy and Safety of Plitidepsin versus Control in 
Immunocompromised Adult Patients with Symptomatic COVID-19 
requiring Hospital Care (NEREIDA) 
 
INVESTIGATIONAL MEDICINAL PRODUCTS: Plitidepsin. 
Protocol Code: AV-APL-B-002-22 
EudraCT No: 2022-002489-34  
 
Version 3.0 - 22 MAR 2023 
Sponsor’s Medical Contact: 
 
 
 
This study will be conducted in compliance with the protocol, Good Clinical Practice (GCP) and 
the applicable regulatory requirements. 
Confidentiality statement 
Information and data included in this protocol contain trade secrets and privileged or 
confidential information which is the property of the Sponsor. No person is authorized to make 
it public without written permission of the Sponsor. These restrictions on disclosure will apply 
equally to all future information supplied to you which is indicated as privileged or confidential. 
This material may be disclosed to and used by your staff and associates as it may be necessary 
to conduct the clinical study. 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 2
 
SPONSOR APPROVAL 
I have read the following and approve it: 
 
 
 
________________________________     ____________________________  
 
      
 
 
Date 
 
 
 
 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 3
 
 
TABLE OF CONTENTS  
LIST OF TABLES ...................................................................................................................................... 6 
LIST OF FIGURES ..................................................................................................................................... 6 
SYNOPSIS ............................................................................................................................................... 7 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS .......................................................................... 29 
1. 
INTRODUCTION .......................................................................................................................... 34 
1.1 
BACKGROUND .................................................................................................................................. 34 
1.2 
INFORMATION ON THE STUDY DRUG .................................................................................................... 36 
1.2.1 
Plitidepsin ........................................................................................................................... 38 
1.2.1.1 
Name and Chemical Information ............................................................................................ 38 
1.2.1.2 
Mechanism of Action .............................................................................................................. 39 
1.2.1.3 
Summary from the Clinical Development of Plitidepsin ......................................................... 40 
1.2.1.3.1 
Benefit-Risk Considerations in COVID-19 ........................................................................... 44 
1.2.1.3.2 
Effects of Plitidepsin in Immune System ............................................................................ 46 
1.2.1.3.3 
Clinical data of plitidepsin in immunocompromised patients with COVID-19 ................... 50 
1.3 
STUDY RATIONALE ............................................................................................................................. 53 
2. 
STUDY OBJECTIVES ..................................................................................................................... 54 
2.1 
PRIMARY OBJECTIVE .......................................................................................................................... 54 
2.2 
KEY SECONDARY OBJECTIVE ................................................................................................................ 54 
2.3 
SECONDARY AND EXPLORATORY OBJECTIVES .......................................................................................... 54 
2.3.1 
Efficacy secondary objectives ............................................................................................. 54 
2.3.2 
Safety secondary objectives ................................................................................................ 54 
2.3.3 
Other secondary objectives................................................................................................. 54 
3. 
OVERALL STUDY DESIGN ............................................................................................................. 55 
3.1 
PRIMARY ENDPOINT .......................................................................................................................... 59 
3.2 
KEY SECONDARY ENDPOINT................................................................................................................. 59 
3.3 
SECONDARY ENDPOINTS ..................................................................................................................... 59 
3.3.1 
Efficacy secondary endpoint ............................................................................................... 59 
3.3.2 
Safety secondary endpoints ................................................................................................ 59 
3.3.3 
Other secondary/exploratory endpoints ............................................................................. 60 
4. 
SELECTION OF PATIENTS ............................................................................................................. 61 
4.1 
INCLUSION CRITERIA .......................................................................................................................... 61 
4.2 
EXCLUSION CRITERIA .......................................................................................................................... 64 
5. 
PLAN OF THE STUDY ................................................................................................................... 65 
5.1 
PLANNED TRIAL PERIODS (INDIVIDUALLY PER PATIENT) ............................................................................. 65 
5.2 
DISCONTINUATIONS ........................................................................................................................... 65 
5.2.1 
Screening Failure ................................................................................................................ 65 
5.2.2 
Treatment Discontinuation ................................................................................................. 65 
5.2.3 
Study Discontinuation ......................................................................................................... 66 
5.2.4 
Lost to follow up. ................................................................................................................ 66 
5.3 
PROTOCOL DEVIATIONS ...................................................................................................................... 67 
5.4 
REPLACEMENT OF PATIENTS ................................................................................................................ 67 
5.5 
PATIENT SCREENING .......................................................................................................................... 68 
5.5.1 
Screening Assessments and procedures ............................................................................. 68 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 4
 
 
 
5.6 
PATIENT RANDOMISATION .................................................................................................................. 70 
5.7 
EVALUATIONS AND PROCEDURES DURING TREATMENT ............................................................................. 71 
5.8 
EVALUATIONS DURING FOLLOW-UP ...................................................................................................... 74 
5.9 
EVALUATIONS AT THE END OF STUDY .................................................................................................... 78 
6. 
TREATMENTS .............................................................................................................................. 80 
6.1 
DESCRIPTION OF TREATMENTS ............................................................................................................. 80 
6.1.1 
Drug Formulation and Supply ............................................................................................. 80 
6.1.2 
Storage conditions .............................................................................................................. 80 
6.1.3 
Administration of Study Medications ................................................................................. 80 
6.1.4 
Treatment Schedule and Dose ............................................................................................ 80 
6.1.5 
Prophylactic Medication ..................................................................................................... 81 
6.2 
DOSE REDUCTION ............................................................................................................................. 81 
6.3 
CONCOMITANT MEDICATION .............................................................................................................. 81 
6.3.1 
Allowed Medications/Therapies ......................................................................................... 81 
6.3.2 
Prohibited Medications/Therapies ..................................................................................... 81 
6.3.3 
Drug-drug Interactions with Plitidepsin .............................................................................. 82 
6.4 
DRUG ACCOUNTABILITY...................................................................................................................... 83 
6.5 
TREATMENT COMPLIANCE .................................................................................................................. 83 
7. 
STUDY EVALUATIONS ................................................................................................................. 83 
7.1 
EFFICACY ......................................................................................................................................... 83 
7.1.1 
Test for COVID-19 ............................................................................................................... 83 
7.1.2 
Chest Imaging ..................................................................................................................... 84 
7.1.3 
Clinical Status...................................................................................................................... 84 
7.1.4 
Other Efficacy Assessments ................................................................................................ 84 
7.2 
SAFETY AND TOLERABILITY ASSESSMENTS .............................................................................................. 85 
7.2.1 
Adverse Event of Special Interest ........................................................................................ 87 
7.3 
ADVERSE EVENTS DEFINITIONS ............................................................................................................ 87 
7.3.1 
Adverse Event (AE) .............................................................................................................. 87 
7.3.2 
Serious Adverse Event (SAE) ............................................................................................... 88 
7.3.3 
Death .................................................................................................................................. 88 
7.3.4 
Life-threatening Event ........................................................................................................ 88 
7.3.5 
Hospitalisation/Prolongation of Hospitalisation ................................................................ 88 
7.3.6 
Unexpected/Unlisted Adverse Event ................................................................................... 89 
7.3.7 
Adverse Reactions ............................................................................................................... 89 
7.3.8 
Serious Adverse Reaction (SAR) .......................................................................................... 89 
7.3.9 
Suspected Unexpected Serious Adverse Reaction (SUSAR) ................................................. 89 
7.3.10 
Adverse Events Related to the Study Drug ..................................................................... 90 
7.3.11 
Expedited Reporting ....................................................................................................... 90 
7.3.12 
Assessment of Causal Relationship to the Study Drug ................................................... 90 
7.4 
ADVERSE EVENTS REPORTING PROCEDURES ........................................................................................... 90 
7.4.1 
Reporting of Adverse Events ............................................................................................... 90 
7.4.2 
Reporting of Serious Adverse Events .................................................................................. 91 
7.4.3 
Reporting Pregnancy Cases Occurred within the Clinical Trial ........................................... 92 
7.5 
ADVERSE EVENTS MONITORING ........................................................................................................... 93 
7.6 
PHARMACOKINETICS .......................................................................................................................... 93 
7.6.1 
Blood Sampling ................................................................................................................... 93 
7.6.2 
Analytical Procedures ......................................................................................................... 94 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 5
 
 
 
7.6.3 
Pharmacokinetic Parameters.............................................................................................. 94 
8. 
STATISTICAL METHODS ............................................................................................................... 94 
8.1 
SAMPLE SIZE CALCULATION ................................................................................................................. 95 
8.2 
ANALYSIS POPULATIONS ..................................................................................................................... 96 
8.2.1 
Full Analysis Set (FAS) Population ....................................................................................... 96 
8.2.2 
Per Protocol Population ...................................................................................................... 97 
8.2.3 
As Treated Population ........................................................................................................ 97 
8.3 
GENERAL CONSIDERATIONS ................................................................................................................. 97 
8.4 
ANALYSIS METHODS .......................................................................................................................... 97 
8.4.1 
Primary Endpoint ................................................................................................................ 97 
8.4.2 
Secondary Endpoints/Other Endpoints ............................................................................... 98 
8.4.3 
Safety Analysis .................................................................................................................... 98 
8.4.4 
Pooled and Subgroup Analyses ........................................................................................... 99 
8.5 
SUMMARY OF ESTIMANDS AND STATISTICAL TESTS ................................................................................... 99 
9. 
ADMINISTRATIVE SECTION ....................................................................................................... 105 
9.1 
ETHICS .......................................................................................................................................... 105 
9.2 
MONITORING, AUDITING AND INSPECTING ........................................................................................... 105 
9.3 
PATIENT INFORMED CONSENT ........................................................................................................... 106 
9.4 
CONFIDENTIALITY/ PATIENTS IDENTIFICATION ....................................................................................... 106 
9.5 
ELECTRONIC CASE REPORT FORMS ..................................................................................................... 107 
9.6 
INSURANCE .................................................................................................................................... 107 
9.7 
RETENTION OF RECORDS .................................................................................................................. 107 
9.8 
USE OF INFORMATION AND PUBLICATION ............................................................................................ 107 
10. 
REFERENCES.............................................................................................................................. 109 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 6
 
 
 
 
LIST OF TABLES 
Table 1 Plitidepsin in vitro activity against different SARS-CoV-2 variants. ............................................ 36 
Table 2 Clinical Studies and Pooled Analysis Supporting the Plitidepsin Clinical Development Program
 ........................................................................................................................................................... 41 
Table 3 Samples for pharmacokinetic evaluations. .................................................................................... 93 
Table 4 Example of simulated posterior probabilities for sample size definition. ...................................... 96 
 
LIST OF FIGURES 
Figure 1 Viral load (top) and histopathology score of inflammation (bottom) in the lung of K18-hACE2 
mouse model, infected with SARS-CoV-2 and treated with plitidepsin. ..........
 
 
Figure 2 Total plasma concentration profiles vs plitidepsin time predicted between 1 hour and 1.5 hour…43 
Figure 3 Visual predictive check describing the time course of plitidepsin plasma in different tumor type.
 ........................................................................................................... 
 
Figure 4 Time variation of lymphocyte counts after the first infusion of treatment (cycle 1/Day 1). .
 
 
Figure 5 Time variation of neutrophil counts after the first infusion of treatment (cycle 1/Day1)......
 
 
Figure 6 Intra-patient Time Variation in Lymphocyte Count per Dose Cohort and Median Trends. ..
 
 
Figure 7 Timeline of principle laboratory and microbiological parameters taking Day 1 of plitidepsin 
therapy as reference, and during hospital admission. ........................................................................ 51 
Figure 8 Timeline of main laboratory and microbiological parameters during and after plitidepsin therapy.
 ........................................................................................................................................................... 52 
Figure 9 Algorithm for Patient Assignment in NEREIDA Basket Trial. ... 
 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 7
 
 
 
SYNOPSIS 
TITLE  
A Multicentre, Open label, Randomised, Controlled, Basket, Pragmatic, 
Phase II, Clinical and Translational Study to Determine the Efficacy and 
Safety of Plitidepsin versus Control in Immunocompromised Adult Patients 
with Symptomatic COVID-19 requiring Hospital Care (NEREIDA). 
PROTOCOL CODE 
AV-APL-B-002-22 
 INVESTIGATORS 
A full list of Investigators will be available as a separate document. 
NUMBER OF SITES / 
TRIAL LOCATION 
More than 50 sites are planned to participate in this study worldwide. 
STUDY OBJECTIVES 
and ENDPOINTS 
Objectives 
Endpoints 
Primary objective 
Efficacy primary objective 
Efficacy primary endpoint 
To evaluate efficacy of 
plitidepsin in pre-specified 
groups of 
immunocompromised 
patients with symptomatic 
COVID-19 requiring hospital 
care vs control in terms of 
mortality. 
1-month† all-cause mortality rate.  
†(Day 30 since randomisation). 
 
Secondary objectives 
Key efficacy secondary 
objective 
Efficacy secondary endpoint 
To compare efficacy of 
plitidepsin vs the control in 
terms of viral clearance, in 
each group. 
Time to confirmed negativisation in 
SARS-CoV-2 antigen test or RT-PCR Ct > 
30. 
 
 
 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 8
 
 
 
Efficacy secondary objectives 
Efficacy secondary endpoints 
To compare efficacy of 
plitidepsin vs the control in 
terms of sustained end of 
hospital care, in each group. 
Time to sustained end of COVID-related 
hospital 
care 
from 
the 
time 
of 
randomisation. Sustained discharge is 
defined as no subsequent admission 
within 30 days of initial end of hospital 
care [up to day 60 (±3)], related to either 
COVID-19 or COVID-19 therapy.   
To compare efficacy of 
plitidepsin vs the control in 
terms of symptomatic 
improvement, in each group. 
Time to sustained improvement (defined 
in Section 7.1.3) and resolution of 
selected COVID-19 signs/symptoms (See 
Appendix 10 - COVID-19 signs/symptoms 
checklist). 
To compare efficacy of 
plitidepsin vs the control in 
terms of clinical status 
(11-category WHO Clinical 
Progression Scale), in each 
group. 
Distribution of patients according to 
their clinical status by the 11-category 
WHO Clinical Progression Scale, at Days 
4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 
(±3) (See Appendix 1 – 11-Point World 
Health Organization (WHO) Clinical 
Progression Scale).  
To compare efficacy of 
plitidepsin vs control in terms 
of the need of any kind of 
supplementary oxygen, in 
each group. 
Percentage of patients requiring oxygen 
therapy on Days 4 (±1), 8 (±1), 15 (±1), 
30 (±2), and 60 (±3). 
Time to sustained discontinuation (i.e., 
at least 7 days) of oxygen 
supplementation. 
 
Safety secondary objectives 
Safety secondary endpoints 
To compare 
safety/tolerability of 
plitidepsin vs the control in 
terms of adverse events, 
adverse reactions and 
mortality, in each group. 
Frequency of the following events (all-
cause and drug-related): 
• Treatment-emergent adverse events 
(TEAEs);  
• TEAEs ≥ grade 3 according to the 
National Cancer Institute [NCI]-
Common Terminology Criteria for 
AEs (CTCAE v.5.0);  


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Protocol Number: AV-APL-B-002-22 
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Protocol. Version 3.0 – 22 March 2023                                                                               Page 9
 
 
 
• Adverse events of special interest 
(AESIs);  
• Serious adverse events (SAEs);  
• Drug-related Serious Adverse Events 
(i.e., SARs); 
• Adverse events leading to treatment 
discontinuation; and  
• Deaths (COVID-19-related/all). 
To compare 
safety/tolerability of 
plitidepsin vs the control in 
terms of abnormal laboratory 
parameters, in each group. 
Change respect to baseline* in individual 
study-defined laboratory parameters 
(See Section 5.7, 5.8 and 5.9 and 
Appendix 8 - Clinical Laboratory 
Analyses). 
To compare 
safety/tolerability of 
plitidepsin versus the control 
in terms of variations of vital 
signs, in each group. 
Change respect to baseline* in individual 
vital signs (See Section 5.7, 5.8 and 5.9). 
 
Other secondary objectives 
To compare efficacy of 
plitidepsin vs the control in 
the need of any type of 
advanced oxygen support 
(high-flow nasal oxygen, 
extracorporeal membrane 
oxygenation (ECMO), or non-
invasive or invasive 
mechanical ventilation), in 
each group. 
Percentage of patients requiring 
advanced oxygen support on Days 4 (±1), 
8 (±1), 15 (±1), 30 (±2,) and 60 (±3). 
Time to intensification of respiratory 
support (WHO >5) (See Appendix 1 – 11-
Point World Health Organization (WHO) 
Clinical Progression Scale). 
Total duration of advanced oxygen 
support. 
To compare efficacy of 
plitidepsin vs the control in 
the need of intensification of 
respiratory or intensive care 
support, in each group. 
Percentage of patients requiring high-
flow oxygen on Days 4 (±1), 8 (±1), 15 
(±1), 30 (±2), and 60 (±3).  
Total duration of high-flow oxygen 
therapy per patient. 
Percentage of patients requiring non-


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 10
 
 
 
invasive mechanical ventilation on Days 
4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 
(±3). 
Total duration of non-invasive 
mechanical ventilation per patient. 
Percentage of patients requiring invasive 
mechanical ventilation or ECMO on Days 
4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 
(±3). 
Total duration of invasive mechanical 
ventilation or ECMO per patient. 
Percentage of patients requiring 
admission to ICU on Days 4 (±1), 8 (±1), 
15 (±1), 30 (±2), and 60 (±3). 
Total duration of intensive care unit 
(ICU) stay. 
To compare efficacy of 
plitidepsin vs the control in 
the need of intensification of 
pharmacological therapies for 
COVID-19, in each group. 
Time to onset of additional (i.e., not 
present at baseline) immune-
modulating drugs. 
Percentage of patients receiving 
immune-modulating drugs on Days 1, 
4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 
(±3). 
Time to onset of additional (i.e., not 
present at baseline) antiviral drugs. 
Percentage of patients receiving 
subsequent antiviral drugs (i.e., not 
present at baseline) on Days 4 (±1), 
8 (±1), 15 (±1), 30 (±2), and 60 (±3). 
To compare efficacy of 
plitidepsin vs control in terms 
of superinfection, in each 
group. 
Percentage of patients with a new 
infection by Days 4 (±1), 8 (±1), 15 (±1), 
30 (±2), and 60 (±3). 
To compare efficacy of 
plitidepsin vs the control in 
terms of all-cause- and 
Cumulative mortality (all-cause and 
related to COVID-19) by Days 4 (±1), 8 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 11
 
 
 
related to COVID-19- 
mortality, in each group. 
(±1), 15 (±1), 30 (±2), and 60 (±3). 
To compare efficacy of 
plitidepsin vs the control in 
terms of subsequent hospital 
admissions, in each group. 
Percentage of patients in each study 
group who require subsequent 
admission within 30 days of initial end 
of hospital care [up to day 60 (±3)], 
related to either COVID-19 or COVID-19 
therapy. 
To compare efficacy of 
plitidepsin vs the control in 
terms of the time course of 
viral load, in each group. 
Change respect to Day 1 in the viral 
load of SARS-CoV-2, measured by RT-
PCR, by Days 4 (±1), 8 (±1), 15 (±1), 30 
(±2), 45 (±2), and 60 (±3), in each study 
group. 
Percentage of patients in each study 
group with undetectable viral load, by 
Days 4 (±1), 8 (±1), 15 (±1), 45 (±2), 30 
(±2), and 60 (±3). 
Time to either undetectable viral load of 
SARS-CoV-2 or > 2 logs reduction respect 
to Day 1. 
To compare efficacy of 
plitidepsin vs the control in 
terms of the evolution of 
inflammatory markers, in 
each group. 
Change respect to baseline* in 
inflammatory biomarkers and 
immunological biomarkers (C- 
reactive protein [CRP], procalcitonin, 
lactate dehydrogenase [LDH], ferritin, 
neutrophil-to-lymphocyte ratio, ALC, 
D-dimer, and multiplex cytokines 
assay, by Day 2 (±1), 4 (±1), 8 (±1), 15 
(±1) and 30 (±2) and 60 (±3). 
To compare efficacy of 
plitidepsin vs the control in 
terms of the immune 
response against SARS-CoV-2, 
in each group. 
Change respect to baseline* in individual 
serological assessments against SARS-
CoV-2, by Days 30 (±2), and 60 (±3). 
Change respect to baseline* in individual 
T-cell response against SARS-CoV-2 by 
Days 30 (±2), and 60 (±3).  
To compare efficacy of 
plitidepsin vs the control in 
Change respect to baseline* to Days 15 
(±1), and 30 (±2) in chest X-ray findings 


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Protocol Number: AV-APL-B-002-22 
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ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 12
 
 
 
terms of radiological 
evolution, in each group. 
(Brixia score, centrally assessed) 
(Appendix 9 – Brixia Score). 
To compare efficacy of 
plitidepsin vs the control in 
terms of restoration of the 
therapy for the underlying 
disease, in each group. 
Percentage of patients requiring 
modification of the therapy (drugs, dose 
or schedule) for the underlying disease. 
Time in which pre-scheduled therapies 
for the control of the underlying disease 
were not able to be administered. 
To compare efficacy and 
safety/tolerability of all 
pooled plitidepsin arms vs all 
control arms in all the 
aforementioned endpoints. 
All the aforementioned endpoints. 
To compare efficacy and 
safety/tolerability between 
plitidepsin arms (across 
different groups). 
All the aforementioned endpoints. 
To explore 
prognostic/predictive factors 
for clinical deterioration or 
mortality or drug response. 
Risk for mortality / clinical deterioration 
according to individual clinical 
characteristics and laboratory 
observations present at baseline*, 
scores of validated scales (e.g., ISARIC-
4C), as well as features related to COVID-
19 [including SARS-CoV-2 variant (if 
available), vaccination status, duration of 
the infection, and inflammation-related 
parameters [neutrophil-to-lymphocyte 
ratio (NLR), protein C reactive, 
interleukine-6 (IL-6) and lymphocytes], 
the underlying disease, and other co-
morbidities.  
Increase pharmacology 
knowledge of plitidepsin. 
Limited-sampling pharmacokinetics 
assessment (See Section 7.6). 
PK-PD analysis. 
 
*Baseline = latest test performed between screening and drug 
administration. 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 13
 
 
 
STUDY DESIGN 
This is a multicentre, randomised, controlled, open-label, pragmatic, phase 2 
basket study to assess the efficacy and safety of plitidepsin in different 
groups of immunocompromised patients with symptomatic coronavirus 
disease 2019 (COVID-19) who require hospital care.  
Approximately 150 patients are foreseen for the study, divided in 4 different 
groups of immunocompromised patients: 
• 
Group 1 – Patients receiving immune-suppression due to 
hematopoietic or organ transplantation. 
• 
Group 2 – Patients receiving B-cell depleting therapies. 
• 
Group 3 – Patients receiving other immune-suppressive 
therapies. 
• 
Group 4* – Other situations with immune deficiencies. 
*Not applicable in Spain 
 
Whenever a patient can be allocated to more than 1 group, the priority will 
be group 1 > group 2 > group 3 > group 4*. Patients will be randomised to 
receive best standard care (BSC) ± other antiviral (if clinically indicated) 
on control arm and BSC + plitidepsin on experimental arm. Group 4* will not 
be controlled. 
 
 
 
 
*Not applicable in Spain


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 14
 
 
 
Stratification:  
• 
Group 1: ISARIC mortality (<9/≥9) | Type of graft (allogeneic 
haematopoietic/lung or intestinal/other). 
• 
Group 2: ISARIC mortality (<9/≥9) | Disease (haematological 
neoplasm/other). 
• 
Group 3: ISARIC mortality (<9/≥9) | Disease (neoplasia/other). 
• 
Group 4*: Not applicable. 
 
*Not applicable in Spain 
Study periods: 
The study will include a Screening period, a Treatment period and a Follow-
up period.  
The Screening period starts once the patient has provided written informed 
consent and ends when the patient is randomised in the study. Parameters 
assessed during this time will serve as the baseline values. The screening 
procedures should be preferably performed within 24 h prior to 
administration of study treatment, allowing a maximum of 48 h prior to 
administration of study treatment if required.  
The Treatment period starts on Day 1 (date of randomisation) to 24 h after 
the last dose of the antiviral therapy. If no antiviral was given in the control 
arm, then the treatment period will finish on day 4, for data management 
purposes. Antiviral treatment must always be started on the randomisation 
date.  
The Follow-up period starts after the end-of-treatment and lasts until the 
end-of-study. 
The End of Study (EOS) is Day 60 (±3), or date of early study termination 
unless ongoing SAEs, if applicable.  
The End of Trial (EOT) will be when the Last Patient Last Visit (LPLV) has 
occurred. 
All patients will receive the best available supportive care as per applicable 
local, institutional, national, supranational COVID-19 treatment guidelines.  
Patients assigned to the experimental arms will receive, in addition to BSC, 
plitidepsin as a 60-min IV infusion, during days 1 to 3.  
Patients randomised to the control arm may receive a regulatory-approved 
antiviral treatment in addition to the BSC. 


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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
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For prevention of infusion-related (i.e., plitidepsin-related) reactions (IRRs), 
allergic reactions and emesis reactions, administration of premedications is 
mandatory in the experimental arms.  
All consenting patients discontinuing early from the study before the EOS 
visit should complete the early termination/withdrawal procedures. 
Independent Data Monitoring Committee (IDMC) 
An IDMC will be established to provide study oversight considering that this 
is a multicentre, randomised study being performed in a population at high 
risk for morbidity and mortality. The IDMC will be established and operated 
in compliance with the FDA Guidance for Industry “Establishment and 
Operation of Clinical Trial Data Monitoring Committees”. 
The IDMC will be composed of individuals external not only to the study 
Sponsor but also to the trial managers (including Sponsor’s and Contract 
Research Organisation’s [CRO] medical monitors), and study investigators, 
and will be comprised of at least 1 clinician specialised in the treatment of 
COVID-19 patients, 1 clinician specialised in assessment of clinical study 
safety issues, and 1 biostatistician specialised in analysis of clinical trials. One 
IDMC member will serve as chair and will prepare minutes of each IDMC 
meeting, which will be provided to the study Sponsor, Sponsor’s medical 
monitor, and CRO project manager only at the end of the study.  
The IDMC will be held before enrolling the first patient on study to discuss 
the protocol and analytic plan, ICF, and plans for IDMC monitoring of study 
safety and effectiveness data. 
The IDMC will have responsibility for: 
• 
Review of all SAEs. 
• 
Review of safety/efficacy trends, such as an accumulating 
number of deaths in the study (i.e., from administration of the 
first dose of study drug on Day 1 through Day 60), to determine 
if there is a difference between plitidepsin and control groups 
and potential impact on study conduct. 
• 
Each group will proceed with per protocol pre-established 
recruitment and follow-up and the IDMC Support Team will 
provide the IDMC Chairperson with unblinded information on 
the number of deaths and SAEs. Upon review of this data, if 
either the difference in the percentage of patients with deaths 
or SAEs Grade >3 is at least 20% higher in any IMP group in 
comparison with the Control Group, IDMC Chairperson will 


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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
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contact the other members of the IDMC to assess the continuity 
of the other groups. 
The IDMC can ask for a temporary halt of the clinical trial at any time to 
better analyse any potential benefit/risk concern. 
Study “Stopping rules” 
The Sponsor may suspend or terminate the study if: 
• 
New toxicological or pharmacological findings or safety issues 
that invalidate the earlier positive benefit-risk assessment occur; 
• 
Significant safety concerns, according to the Sponsor’s criteria, 
arise during the conduct of the study; 
• 
The development of plitidepsin is discontinued; 
• 
Any other reason (e.g., best interest of the patients, accrual rate 
makes the completion of the study feasible, etc.) leads to this 
suspension or termination. 
STUDY 
POPULATION 
Immunocompromised adult patients with symptomatic COVID-19 requiring 
hospital care. 
STUDY 
POPULATION 
Inclusion criteria 
To be eligible for inclusion into this study, each patient must fulfil the 
following inclusion criteria within 48 hours prior to randomisation: 
General inclusion criteria: 
1. Signed informed consent obtained prior to initiation of any study-
specific procedures and study treatment; 
2. Patient aged ≥18 years; 
3. Patient diagnosed COVID-19, with the following characteristics:  
a) A regulatory approved test, collected no more than 3 days prior to 
study randomisation, with either a Ct value ≤30 or a positive antigen 
test; 
b) Presence of any of the selected signs/symptom listed in Appendix 
10 - COVID-19 signs/symptoms checklist within the last 24 h; 
4. Patient already admitted or requiring hospital care* for symptomatic 
COVID-19, for which at least one antiviral has failed†,  or cannot be 
used‡, after a minimum washout period of 24h for small molecules 
(e.g., remdesivir, molnupiravir, nirmaltrevir/ritonavir) and 5 days for 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 17
 
 
 
antiviral monoclonal antibodies (e.g., tixagevimab + cilgavimab) or 
convalescent plasma. 
*The definition of hospital care is based on the need to use a 
hospital environment (hospital ward, day hospital) for treatment 
administration and/or clinical monitoring of the patient with 
COVID-19.  
†Failure of a prior antiviral is defined as a documented lack of 
clinical response, plus evidence of persisting positivity for SARS-
CoV-2 in an appropriate biological sample, determined by a 
regulatory approved test, collected no more than 3 days prior to 
study randomisation, with either a Ct value ≤ 30 (RT-PCR) or a 
positive antigen test.  
‡Contraindication, absence of labelled indication, guidelines or drug 
unavailability.    
5. Adequate bone marrow, liver, kidney, and metabolic function, 
defined by the following tests performed at local laboratory: 
o Absolute neutrophil count ≥500/mm3 (0.5 x 109/L); 
o Platelet count ≥ 50 000/mm3 (50 x 109/L); 
o Alanine transaminase (ALT) ≤3 x upper limit of normal (ULN) 
(≤5 x ULN if pre-existent liver involvement by the underlying 
disease); 
o Serum bilirubin ≤1.5 x ULN (or direct bilirubin <1.5 x ULN 
when total bilirubin is above ULN); 
o Estimated glomerular filtration rate ≥30 mL/min [CKD-EPI 
Creatinine Equation (2021)].   
6. Females of childbearing potential must have a negative serum or urine 
pregnancy test by local laboratory at study enrolment and must be non-
lactating. 
7. Females of child-bearing potential must use highly effective 
contraceptive methods, while on study treatment and for 6 months after 
last dose of plitidepsin. Fertile males with partners of child-bearing 
potential must use effective contraception, while on study treatment 
and for 6 months after last dose of plitidepsin (See Appendix 2 – 
Contraception and pregnancy testing). Patients in the control arm must 
follow contraception methods indicated in the approved product 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 18
 
 
 
information (summary of product characteristics [SmPC] or leaflet). If no 
information is available in the approved product information, patients in 
the control arm must use highly effective contraception (females of 
child-bearing potential) and effective (fertile males with partners of 
child-bearing potential) for at least one week after the study completion 
or the time indicated based on the investigator’s discretion.  
Group-specific inclusion criteria: 
• 
Group 1 – Patients receiving, within the last 30 days, immune-
suppressive 
therapy 
due 
to 
haematopoietic 
or 
organ 
transplantation. 
o Haematopoietic transplantation. 
o Solid Organ Transplantation: 
▪ 
Lung / intestinal. 
▪ 
Other. 
• 
Group 2 – Patients receiving B-cell depleting therapies within the 
last 6 months*. 
Includes (but is not limited to): 
o Monoclonal antibodies (mAbs) targeting CD19, CD20, CD38, 
or CD52 (e.g., rituximab, ocrelizumab, ofatumumab, 
daratumumab, alemtuzumab). 
o B-cell activating factor (BAFF) inhibitors (e.g., belimumab). 
o Bruton's tyrosine kinase (BTK) inhibitors (e.g., evobrutinib, 
ibrutinib). 
o Chimeric antigen receptor T cell therapy (CAR-T) (e.g., anti-
CD19 CAR-T cell). 
*Time restriction is not applicable for CAR-T cell therapy. 
• 
Group 3 – Patients receiving, within the last 30 days, other 
immune-suppressive therapies. 
o Other immunosuppressive therapies not including B-cell 
depleting agents for the treatment of auto-immune 
disorders. 
o Chemotherapy 
or 
targeted 
therapies 
with 
immunosuppressive potential for solid tumours or 
haematological disorders.  
o Chronic glucocorticoids (i.e., equivalent to prednisone ≥ 
20 mg/day for more than 1 month). 
 
• 
Group 4* – Other situations with immunodeficiency. 
Includes (but is not limited to): 
o Primary immune deficiencies. 


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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
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o Human immunodeficiency virus (HIV) infection, with CD4+ T 
lymphocytes < 200 cells/µL in the last month.  
o Radiation therapy within the last 3 months- requires 
documentation of ALC < 500 cells/µL. 
o Haematological neoplasia or myelodysplasia not currently 
receiving any therapy. 
o Other situations with a documentation of ALC < 500 cells/µL. 
*Not applicable in Spain 
Further information about groups and immunosuppressive 
therapies can be found in Appendix 12 – Definition of NEREIDA Study 
Groups and lists of immunosuppressive treatments. 
Exclusion criteria 
1. Evidence of critical illness, defined by at least one of the following:  
• 
Respiratory failure defined based on resource utilization 
requiring at least one of the following: endotracheal intubation 
and mechanical ventilation, ECMO, or clinical diagnosis of severe 
acute respiratory distress syndrome with PaO2*/FiO2 ≤ 100.  
*In case a direct measure of PaO2 has not been obtained, it 
should be imputed according to a referenced formula (Ellis or 
Rice) (Appendix 5 - Imputation of PaO2/FiO2 ratio). For sites 
located over 1000 m altitude above sea level, PaO2/FiO2 ratio 
will be adjusted (Appendix 6- Adjustment of PaO2 from a Site at 
High Altitude; See also Appendix 7 for FiO2 imputations from 
oxygen flow rates). 
• 
Shock requiring vasopressors. 
• 
Multi-organ dysfunction/failure. 
2. (Criterion eliminated and merged with inclusion criterion #4, based 
on AEMPS recommendations). 
3. Any of the following cardiac conditions or risk factors: 
• 
Cardiac infarction or cardiac surgery episode within the last 
month;  
• 
History of known congenital QT prolongation; 
• 
Known structural cardiomyopathy with abnormal LVEF (<50%); 
• 
Current clinical evidence of heart failure or acute cardiac 
ischaemia (New York Heart Association (NYHA) class III-IV).           


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
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4. Hypersensitivity to the active ingredient or any of the excipients 
(mannitol, macrogolglycerol hydroxystearate, and ethanol) or 
contraindication to receive dexamethasone, antihistamine H1/H2, 
or anti-serotoninergic 5HT3 agents.  
5. Females who are pregnant or breast-feeding. 
6. Females and males with partners of childbearing potential (females 
who are not surgically sterile or postmenopausal defined as 
amenorrhoea for >12 months) who are not using at least 
1 protocol-specified method of contraception. 
7. Any situation currently requiring increasing needs of immune 
suppressive agents (e.g., acute graft rejection, flare of autoimmune 
disorder, or cytokine storm syndrome). 
8. Any other clinically significant medical condition (including major 
surgery within the last 3 weeks before screening) or laboratory 
abnormality that, in the opinion of the investigator, would 
jeopardise the safety of the patient or potentially impact on patient 
compliance or the safety/efficacy observations in the study. 
9. Participation in another clinical study involving an investigational 
drug within 30 days prior to screening. 
 
EXPECTED NUMBER 
OF PATIENTS 
Approximately 150 are foreseen to be enrolled in the study. 
REPLACEMENT OF 
PATIENTS 
Patients will not be replaced, but accrual in each group will continue until 
the target sample size of patients evaluable for the primary endpoint is 
reached. 
STUDY DRUGS 
FORMULATION 
Powder for concentrate for solution for infusion. 
ROUTE OF 
ADMINISTRATION 
Intravenous (IV) infusion. 
TREATMENT 
SCHEDULE AND 
DOSE 
Plitidepsin will be administered as a 60-min (±5 min) IV infusion, every 24 h 
(±30 min) for 3 consecutive days, at a dose of 2.5 mg. 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 21
 
 
 
PROPHYLACTIC 
MEDICATION 
For prevention of plitidepsin related infusion reactions, the following 
premedications MUST be given to each patient and should be completed no 
more than ~2 hours to 20 minutes before initiating each plitidepsin infusion: 
• 
Dexamethasone phosphate* 8 mg IV (equivalent to 6.6 mg of 
dexamethasone base); 
• 
Dexchlorpheniramine maleate* 5 mg IV (or diphenhydramine 
hydrochloride 25 mg IV); 
• 
Famotidine* 20 mg IV (or equivalent such as ranitidine 50 mg IV). 
Oral famotidine is acceptable, but in this case the dose should be 
40 mg and it must be administered 2 hours (±15 min) before 
plitidepsin; 
• 
Palonosetron* 0.25 mg IV (tropisetron 5 mg IV could be considered 
if palonosetron is not available. It should be administered orally 
(PO)/IV on Days 4 and 5 if tropisetron 5 mg was administered on 
Days 1, 2 and 3). 
*Provided by the Sponsor, who is also in charge of the labeling (except when 
the site uses its own medication for the trial). 
 
ALLOWED 
MEDICATIONS/ 
THERAPIES 
 
Any medications, with the exceptions noted below and in Section 6.3.2 , 
which are considered necessary for the patient’s welfare, and which will not 
interfere with the study medication, may be given at the discretion of the 
Investigator. Medications which MUST be administered as part of the study 
treatment schedule are listed in Section 6.1.5.  
Administration of all concomitant drugs received by the patient during the 
study must be reported in the appropriate section of the eCRF along with 
dosage information, dates of administration and reasons for use. 
Additionally, any unplanned diagnostic, therapeutic or surgical procedure 
performed during the study period must be recorded in the eCRF.  
PROHIBITED 
MEDICATIONS/ 
THERAPIES 
 
• 
Other investigational agents. 
• 
Concomitant use of plitidepsin and antiviral treatments for COVID-
19. 
• 
Strong CYP3A4 inhibitors and inducers throughout plitidepsin 
treatment period and until 24-h washout period (See Appendix 3 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 22
 
 
 
Inhibitors and Inducers of CYP3A4), unless medical judgement that 
they are for the patient’s best interest.  
 
Investigator may decide to stop the study treatment at any time after the 
first dose of plitidepsin and initiate other pharmacological treatments for 
COVID-19.  
 
DRUG-DRUG 
INTERACTIONS 
WITH PLITIDEPSIN 
Interactions with CYP3A4 inhibitors and inducers: In vitro studies indicate 
that CYP3A4 is the major enzyme involved in plitidepsin metabolism, 
followed by CYP2A6 and CYP2E1. However, a pharmacokinetic population 
analysis of plitidepsin including 283 patients indicated that concomitant 
administration of CYP3A4 inhibitors and inducers does not affect exposure 
to plitidepsin. Nevertheless, it cannot be ruled out that concomitant 
treatments that strongly inhibit or induce CYP3A4 may modify the efficacy 
and/or increase the probability of side effects associated with plitidepsin, so 
they should be avoided. 
• 
Co-administration with CYP3A4 strong inhibitors and inducers 
should be avoided, as they may affect the plasma concentration of 
plitidepsin, unless medical judgement that they are for the patient’s 
best interest. 
• 
Co-administration with CYP3A4 moderate inhibitors and inducers 
should be used with caution, as an effect on plitidepsin exposure 
cannot be excluded. 
A list of CYP3A4 inducers and inhibitors is included in Appendix 3 - Inhibitors 
and Inducers of CYP3A4. 
DOSE REDUCTION 
No dose modifications are foreseen at the experimental arm. 
SAFETY 
EVALUATION 
CRITERIA 
Safety parameters/covariates include: 
• 
Demographic Data including gender and age; 
• 
Medical history and prior medications and procedures; 
• 
ISARIC 4C Mortality & 4C Deterioration scores (Appendix 4); 
• AEs will be monitored throughout the study, which will start 
immediately after the patient Informed Consent Form (ICF) is signed; 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
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CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 23
 
 
 
• 
Pregnancy; 
• 
Concomitant medications and procedures; 
• 
Physical examination including height and weight. Body Mass Index 
will be calculated; 
• 
Vital 
signs 
include 
body 
temperature (°C), 
systolic 
blood 
pressure (SBP) (mmHg), diastolic blood pressure (DBP) (mmHg), 
pulse rate (bpm) and respiratory rate (breaths/min); 
• 
Laboratory 
tests 
include 
haematology, 
coagulation 
and 
biochemistry; 
• 
Urinalysis; 
• 
Electrocardiograms (ECGs) include overall assessment, heart rate 
(HR), PR interval (PR), QRS duration (QRS), uncorrected QT interval 
(QT), and corrected QT interval by Fredericia (QTcF), and will be 
performed at the study site. 
EFFICACY 
EVALUATION 
CRITERIA 
Efficacy parameters include: 
• 
Mortality (all-cause and related to COVID-19); 
• 
COVID-19 signs and symptoms will be collected daily from Day 1 until 
the end of hospital care; thereafter at Days 4, 8 (±1), 15 (±1), 30 (±2), 
45 (±2), and 60 (±3); 
• 
Clinical status is assessed using the WHO 11-point Clinical 
Progression Scale; 
• 
Lung function tests include fraction of inspired oxygen (FiO2) (%), 
oxygen saturation (SpO2), partial pressure of oxygen (PaO2) (mmHg) 
[could be imputed from oxygen saturation (SpO2)]. PaO2/FiO2 ratio 
will also be calculated (See Appendix 5 - Imputation of PaO2/FiO2 
ratio);  
• 
SARS-CoV-2 quantitative real-time-reverse transcription polymerase 
chain reaction test (qRT-PCR) will be performed by central laboratory 
from oro-nasopharyngeal exudates on Day 1 before administration 
of the study drug and repeated at Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), 
45 (±2), and 60 (±3). Name of the variant will be collected if known; 
• 
SARS-CoV-2 Rapid Antigen test (RAT) will be performed every other 
day from Day 4 until discharge, and always at Days 1, 4 (±1), 8 (±1), 
15 (±1), 30 (±2), 45 (±2), and 60 (±3); if RAT is positive at discharge, 
RAT should also be monitored on a weekly basis until negative. A 


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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 24
 
 
 
negative result should be confirmed with a second RAT within 48 h, 
by the investigator team;  
• 
Humoral response anti-SARS-CoV-2; 
• 
T-cell response against SARS-CoV-2; 
• 
Additional therapies for COVID-19; 
• 
Type and dates of oxygen support; 
• 
Radiological scores (chest X-ray, Brixia score; centrally assessed) (See 
Appendix 9 – Brixia Score); 
• 
Death date and reason; 
• 
Dates of discharge from ICU or hospital, and date of eventual re-
admission; 
• 
Inflammatory and immunological biomarkers, including C-reactive 
protein (CRP), procalcitonin, lactate dehydrogenase (LDH), ferritin, 
neutrophil-to-lymphocyte ratio, ALC, D-Dimer, and multiplex 
cytokines assay;  
• 
Compliance with scheduled therapies for the underlying disease. 
PHARMACOKINETIC 
(PK) EVALUATIONS 
PK analysis of plitidepsin concentration-time data will be performed in 30 
patients using non-linear mixed-effects modelling. Individual PK parameters 
will be (See Section 7.6): 
• 
Plasma total area under the curve (AUC). 
• 
Plasma total clearance (CL). 
 
STATISTICAL 
METHODS 
This is an exploratory basket study to adequately approach the 
heterogeneity of clinical conditions included under the concept of immune 
compromise. The study  implements a Bayesian methodology. This decision 
has been adopted after taking into account the relative low frequency of the 
conditions under study, the relative absence of prospective data of the 
outcomes of COVID-19 for the study populations (most of the available data 
are retrospective and belong to the early waves of the pandemic, with 
different viral variants than nowadays), along with the changing medical 
practices in these settings. Under these premises, a Bayesian approach is 
more appropriate in terms of efficient decision-making process and will 


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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 25
 
 
 
provide novel information for future therapeutic development in these 
clinical conditions. 
The trial will use a minimallyinformative prior of N(0,1) to update the 
information provided by the trial data to produce a posterior distribution. 
The resultant posterior distribution will serve as a starting point for further 
trials within this area, whether as a key part of any information data packs 
provided to experts ahead of an elicitation meeting, or directly as the 
informed prior or hypotheses for further trials.  
A calibrated Bayesian hierarchical model (CBHM) described by Chu and Yuan 
will be used to analyse the data, allowing data to be analysed by group, and 
results from each group will be used to enrich the data of the other groups. 
The calibration of the model allows strong information sharing when the 
treatment effect is similar among baskets and shrinks that sharing towards 0 
as the treatment effect becomes more heterogeneous. 
An overall type I error of 5% will be used to analyse by means of credible 
regions the OR against a null of OR=1 [log(OR)=0], the case where no 
discernible difference is induced from intervention. 
Simulation was used to perform sensitivity analysis on a range of informed 
priors against a minimally informative prior to ensure that its use would not 
impact the results on the data too far.  
The statistical methods for this study are described in a detailed statistical 
analysis plan (SAP). 
SAMPLE SIZE 
A) Randomised groups (1-3) 
Patients in three of the four groups will be randomly assigned to one of two 
arms. Each randomised group will have a sample size of 40 patients (20 in 
the experimental arm, 20 in the control arm). 
Within the CBHM simulations, 5000 Markov-chain Monte Carlo (MCMC) 
repetitions per scenario were simulated with a range of operating 
characteristics for the null, alternative and true mortality rates. A maximum 
sample size of 40 per randomised group was set through the CBHM 
simulations, which in turn determines whether there is a need for the total 
sample size to be allocated. These simulations cover mortality rates between 
5 and 95%. Within all simulations, the full 120 patients were used, suggesting 
that this is the minimum sample size that enables decision-making.  As an 
example, if the mortality rate in each group is 20% and we target a clinically 
meaningful reduction of 12.5% in the experimental arm (H0=20%, H1=7.5%), 
and the assumed true mortality rate is 7.5%, the simulation demonstrates 


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Protocol Number: AV-APL-B-002-22 
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ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 26
 
 
 
that a sample size of 40 per basket will allow for a posterior probability 
around 0.9 in each basket (i.e. 90% probability that the experimental arm is 
better than the control in each basket), ranging from 0.87 (when the data 
from the fastest basket is analysed) to 0.915 (at the end of the accrual of the 
last basket) (type I error rate= 0.05). 
 
B)  Non-controlled group 4* 
Many different clinical conditions cannot be classified into one of the 3 
randomised groups and have been pooled into a fourth group of 30 patients. 
As there is a remarkable intragroup heterogeneity, this group will not be 
controlled and will contribute to the objective of increasing safety 
knowledge of plitidepsin. . A Fleming’s Phase II procedure with a 1-stage 
design has been used to estimate the sample size needed to rule out that 
plitidepsin induces drug-related SAEs in at least 35% of the patients (null 
hypothesis), and to confirm that the respective rate is 10 % or lower 
(alternative hypothesis), with a type I error of 0.05 and a power of 90%.  
If at least 25 patients out of a total sample of 30 do not experience a drug-
related SAE, the null hypothesis will be rejected. 
*Not applicable in Spain 
METHODS OF ANALYSIS 
Analysis of the primary and other time-to-event efficacy endpoints will be 
performed primarily in the FAS but also in the “Per Protocol” and “As 
treated” populations. Supportive analyses will be performed on the Per 
Protocol population. Safety endpoints analyses will be based on the “As 
treated” Population. 
Primary endpoint 
The difference between treatment and control arms in all-cause mortality at 
30 days will be examined using a Bayesian comparison of proportions within 
the calibrated Bayesian hierarchical model (CBHM) framework described by 
Chu and Yuan. The use of the CBHM allows for flexible information borrowing 
across all groups, with strong borrowing in the case when treatment effect 
is homogeneous among the groups and less borrowing as the effect becomes 
more heterogeneous.  
In this framework the odds ratio is modelled as θi=log(oddsi) with a normal 
distribution of unknown mean (μ) and variance (σ2). The prior distribution 
used by the framework will be a minimally informative prior using N(0.1). 
The variance (σ2) represents the degree of heterogeneity between the 
patient groups, with 0 representing the case where there is homogeneity of 


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Protocol Number: AV-APL-B-002-22 
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ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 27
 
 
 
treatment effect and thus complete pooling of the results across patient 
groups, with adjustment for targeted p1 rates in each group, whereas a value 
closer to infinity would lead to no borrowing across groups. These estimates 
are iteratively updated within the modelling procedure based on the 
available data. 
As each basket is completed, they will be evaluated for efficacy, with 
information gained being used to update the analyses of successive baskets.  
Once all patients recruited to a basket have completed the trial, a formal 
analysis will be performed on the patients within the basket. The result will 
be able to stand on its own for the specific subpopulation, and the 
information from this analysis will be used to enrich the information at 
analysis of the proceeding baskets. 
Results in later baskets will be related to pooled data, however if one basket 
is not seen to be homogeneous to other baskets, the information from that 
basket can be run without the pooled information from other baskets and its 
information not pulled through into later analyses. 
As a sensitivity analysis the Bayesian hierarchical model will be used.  
The cut-off for considering further clinical development of plitidepsin in each 
randomised group will be a posterior probability of superiority for the 
experimental arm equal to or greater than 65%.  
Secondary endpoints/Other endpoints 
Frequency tables will be prepared for categorical variables, and continuous 
variables will be described by means of summary tables, which will include 
the median, mean, standard deviation, interquartile range, minimum, and 
maximum of each variable. 
In addition to Bayesian methods, frequentist methods will be also calculated. 
Counts and percentages, with their corresponding exact confidence 
intervals, will be calculated for the binomial endpoints. Time-to-event 
variables and their set time estimates will be analysed according to the 
Kaplan-Meier method and Cox regressions. 
Safety analyses will include AEs, SAEs, deaths, laboratory evaluations and 
study drug discontinuations due to AEs will be tabulated in a descriptive way.  
Pooled and subgroup analyses will be performed, taking into consideration 
baseline characteristics of the patients and of the underlying disease, risks 
factors for clinical deterioration or death, vaccination status,duration of the 
infection prior to study entry, and concomitant therapies (e.g., 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 28
 
 
 
immunomodulatory 
agents 
—including 
glucocorticoids—, 
oxygen 
supplementation). 
DURATION OF 
STUDY PERIOD (per 
patient) 
 
 
 
 
 
• 
Screening period: From the day of ICF signing prior to randomisation). 
• 
Day 1: Date of randomisation. Treatment must be started on the same 
date.  
• 
Treatment period: from Day 1 to 24 h after the last dose of the 
antiviral therapy; if no antiviral was given in the control arm, then the 
treatment period will finish on Day 4, for data management purposes. 
• 
Follow-up period: From the end-of-treatment to end-of-study. 
• 
EOS: Day 60 (±3) or date of early study termination unless ongoing 
SAEs, if applicable.  
• 
Overall study duration: 60 (±3) days since randomisation. 
 


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Protocol. Version 2.0 – 22 March 2023                                                                               Page 29
 
 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
 
5-HT3 
Serotonin 
AE(s) 
Adverse Event(s) 
AEMPS 
AESI 
ALC 
ALI 
Spanish Agency of Medicines and Medical Devices 
Adverse Events of Special Interest 
Absolute Lymphocyte Count 
Acute Lung Injury 
ALT 
Alanine Aminotransferase 
ANC  
Absolute Neutrophil Count 
AP 
APTT 
ARDS 
Alkaline Phosphatase 
Activated Partial Thromboplastin Time 
Acute Respiratory Distress Syndrome 
AST 
AUC 
BAFF 
hCG 
bpm 
BSC 
BTK 
BUN 
CAR-T 
CDHM 
CC50 
CCL 
CD 
Aspartate Aminotransferase 
Area Under the Curve 
B-cell Activating Factor 
beta Human Chorionic Gonadotropin 
Beats per minute 
Best Standard Care 
Bruton’s Tyrosine Kinase 
Blood Urea Nitrogen 
Chimeric Antigen Receptor T-cell Therapy 
Calibrated Bayesian Hierarchical Model 
50% of maximum cytotoxic effect  
Chronic Lymphocytic Leukemia 
Cluster of Differentation 
CI 
CL 
Confidence Interval 
Clearance 
COVID-19 
Coronavirus Disease 2019 
CPK 
Creatine Phosphokinase 
CrCL 
CRISPR 
CRO 
CRP 
CRX  
Creatinine Clearance 
Clustered Regularly Interspaced Short Palindromic Repeat 
Contract Research Organisation 
C-reactive Protein 
Chest X-ray 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 30
 
 
 
Ct 
CTCAE 
CTFG 
CYP 
D 
DMVs 
dsRNA 
EC 
Cycle Threshold 
Common Terminology Criteria for Adverse Events 
Clinical Trial Facilitation Group 
Cytochrome P450 
Day 
Double-membrane Vesicle(s) 
Double-stranded Ribonucleic Acid 
Ethics Committee 
ECG 
Electrocardiogram 
ECMO 
Extracorporeal Membrane Oxygenation 
e-CRF 
eEF1A 
e.g. 
Electronic Case Report Form 
Eukaryotic Elongation Factor 1A 
For example, from the latin exempli gratia 
EOS 
ET 
Etc. 
EUA 
F 
FAS 
End of Study 
Early Termination 
Etcetera 
Emergency Use Authorisation 
Female 
Full Analysis Set 
FDA 
FiO2 
FLIM-FRET 
g 
U.S. Food and Drug Administration 
Fraction of Inspired Oxygen 
Fluorescence Lifetime Imaging-Fluorescence Resonance Energy Transfer 
Grams 
GCP 
Good Clinical Practice 
GGT 
Gamma-glutamyltransferase 
H 
hACE2 
HCoV 
HCT 
HCV 
HEK 
HIV 
Hours 
Human Angiotensin converting Enzyme 2 
Human Coronavirus 227E 
Haematopoietic Cell Transplantation 
Hepatitis C Virus 
Human Embryonic Kidney (cell) 
Human Immunodeficiency Virus 
HR 
Heart Rate 
IB 
Investigator’s Brochure 
IC50 
IC90 
Half (50%) Maximal Inhibitory Concentration  
90% Maximal Inhibitory Concentration 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 31
 
 
 
ICF 
Informed Consent Form 
ICH 
ICU 
International Conference on Harmonization 
Intensive Care Unit 
IDMC 
i.e. 
Independent Data Monitoring Committee 
That is, from latin id est 
IEC 
Independent Ethics Committees 
IL 
Interleukin 
IMP 
IMV 
INN 
INR 
Investigational Medicinal Product 
Invasive Mechanical Ventilation 
International Nonproprietary Names 
International Normalised Ratio 
IRB 
Institutional Review Board 
IRR 
IUD 
IUS 
ISARIC-4C 
Infusion-related Reactions 
Intrauterine Device 
Intrauterine Hormone-releasing System 
International Severe Acute Respiratory and Emerging Infection Consortium-
Coronavirus Clinical Characterisation Consortium 
ITT 
Intention-to-treat 
IUPAC 
International Union of Pure and Applied Chemistry 
IV 
K18 
L 
LBR 
Intravenous  
Human Keratin 18 
Liters 
Lung-to-Blood Ratio 
LDH 
LLN 
Log 
LoQ 
LVEF 
MAbs 
MCMC 
Lactate Dehydrogenase 
Lower Limit of Normal 
Logarithm 
Limit of Quantification 
Left Ventricular Ejection Fraction 
Monoclonal Antibodies 
Markov Chain Monte Carlo 
MedDRA 
M 
M 
Medical Dictionary for Regulatory Activities 
Meter 
Male 
mg 
Milligram 
Min 
mL 
Minutes 
Milliliter(s) 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 32
 
 
 
µL 
mm 
MM 
mmHg 
Microliters 
Millimeters 
Multiple Myeloma 
Millimeter(s) of Mercury 
N 
Nucleocapsid/Number 
NCI 
National Cancer Institute 
NCI-CTCAE 
National Cancer Institute Common Terminology Criteria for Adverse Events 
NIV 
nM 
Non-invasive Ventilation 
Nanomolar 
NT-pro-BNP 
NYHA 
N-terminal Pro-B-type Natriuretic Peptide 
New York Heart Association 
PaO2 
PaO2/FiO2 
Pbar 
P-gp 
Partial Pressure of Oxygen 
Partial Pressure of Oxygen Fraction of Inspired Oxygen 
Barometric Pressure 
P-glycoprotein 
PhV 
PI 
Pharmacovigilance 
Principal Investigator 
PK 
Pharmacokinetic(s) 
PK/PD 
PO 
PO2 
POC 
PR 
Pharmacokinetic/Pharmacodynamic 
Oral Administration 
Partial Pressure of Oxygen 
Proof of Concept 
Interval PR 
q4wk 
Q4 
qPCR 
Every four Weeks 
Quarter 4 
Quantitative Polymerase Chain Reaction 
qRT-PCR 
QRS 
QT 
QTcF 
qw 
RAT 
RBC 
Quantitative Reverse Transcription Polymerase Chain Reaction 
Interval QRS 
QT interval 
QT Interval Corrected using Fredericia’s Formula 
Every Week 
Rapid Antigen Test 
Red Blood Cell 
RNA 
Ribonucleic Acid 
RT-PCR 
Real Time Polymerase Chain Reaction 
SAE(s) 
Serious Adverse Event(s) 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 33
 
 
 
SAP 
SARs 
SARS-CoV 
Statistical Analysis Plan 
Serious Adverse Reaction(s) 
Severe Acute Respiratory Syndrome Coronavirus 
SARS-CoV-2 
SBP 
Severe Acute Respiratory Syndrome Coronavirus 2 
Systolic Blood Pressure 
SOT 
Solid Organ Transplantation 
SmPC 
Summary of Product Characteristics 
SpO2 
SUSAR 
TEAE 
TNF 
Saturation of Oxygen 
Suspected Unexpected Serious Adverse Reaction 
Treatment-emergent Adverse Event 
Tumour Necrosis Factor alpha 
ULN 
vs 
Upper Limit of Normal 
versus 
WBC 
White Blood Cells 
WHO 
World Health Organization 
Wk 
WMA 
Week(s) 
World Medical Association 
WOCBP 
wt 
Women Of Childbearing Potential 
Wild-type 
Y 
Yes 
 
 
 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 34
 
 
 
1. INTRODUCTION 
1.1 
BACKGROUND 
Coronavirus disease 2019 (COVID-19), produced by the severe acute respiratory 
syndrome coronavirus type 2 (SARS-CoV-2), has caused a global pandemic that was 
initially reported in China in December 2019 (1-3). The clinical manifestations of COVID-
19 range from an asymptomatic course to mild (low-level fever, fatigue, and dry cough 
along with symptoms such as nasal congestion, a runny nose, diarrhea and slight 
weakness with or without pneumonia) to the severe form, characterized by dyspnoea 
and/or hypoxemia that can rapidly progress to septic shock, irreversible metabolic 
acidosis, coagulation disorders, hospital admission, need for intensive care and death in 
4.3-15% of patients (3-6). According to the World Health Organization (WHO), as of June 
2022, more than 535 million cases of COVID-19 have been reported in the world with 
more than 6.3 million deaths (7). 
Immunocompromised patients are at increased risk for severe and fatal coronavirus 
disease 2019 (COVID-19) outcomes. In a multicentre retrospective study of over 400 
solid organ transplant (SOT) recipients with COVID-19, 78% required hospitalisation, 
34% required intensive care, and 27% required mechanical ventilation (8). 
Haematopoietic cell transplant (HCT) recipients also experience high rates of COVID-19–
related complications, with up to 15% of patients requiring intubation (9). Additionally, 
some studies have shown that mortality rates of SOT and HCT recipients with COVID-19 
range between 20% and 30% (8, 9), although more recent studies of SOT recipients, 
including a study showing comparable outcomes between SOT recipients and non-SOT 
controls (10, 11), have shown lower mortality rates (4.4%–9.6%) (12, 13). Patients with 
solid tumors, human immunodeficiency virus (HIV), and primary immunodeficiencies 
and COVID-19 are also at high risk for severe outcomes, intubation, and death (12, 13). 
Multiple studies have also demonstrated that vulnerable populations such as patients 
at increased age with comorbidities are at increased risk of severe COVID- 19 disease 
and death (13-18). However, data on the outcome of patients with haematological 
malignancies, especially in the field of malignant lymphoma in patients with COVID-19, 
are still limited. Lymphoma patients, who are often already immunosuppresed by their 
disease per se, frequently receive lymphodepleting therapies including anti-CD20 
monoclonal antibodies, chemotherapy, and other targeted agents also leading to an 
additional immunosuppressive effect (19). The speed and degree of humoral and 
cellular immune recovery following prolonged immunosuppression after therapy 
interruption is also unclear. Futhermore, the impact of different therapeutic approaches 
in patients with COVID-19 remains poorly defined, but active chemotherapy appears to 
be associated with increased risk of death in cancer patients with COVID-19 (20). A 
paradigmatic frame of the high deterioration and mortality risk is sustained by a recent 
retrospective, multicentre, international study of the data from 63 mantle cell 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 35
 
 
 
lymphoma (MCL) patients with a median age of 64 years (44-84) with evidence of a 
COVID-19 infection. The analysis demonstrated a frequent fatal outcome (44.4%), 
especially in patients with need for hospitalisation (61%) or intensive care (94%) and 
undergoing current or recent rituximab therapy (21). The negative impact of SARS-CoV-
2 infection in patients with haematological malignancies is also illustrated by a UK-wide 
retrospective study in relapsed/refractory myeloma (RRMM) patients (n=107) 
undergoing anti-CD38-based therapy that showed a 159-days cumulative duration of 
infection-related hospitalisation (22). In line with this, a recent analysis of a 
retrospective observational study on 593 patients with haematological malignancies 
included in the EPICOVIDEHA registry suggest that infection with SARS-CoV-2 Omicron 
variants was associated with considerable morbidity and mortality (23). In fact, the 
mortality among hospitalised patients with haematological malignancies was found to 
be 16.5% (23) which is lower than during the COVID-19 waves of 2020 and 2021, but 
considerable higher than previously reported mortality rates in immunocompetent 
patients with omicron infections (24-26), and in agreement with a small recent 
preliminary report on omicron in chronic lymphoid leukemia patients, where 23% 30-
day mortality was reported (27). Furthermore, data on serologic response in patients 
with haematologic malignancies following COVID-19 vaccination are limited. Recent 
systematic reviews found a correlation between active cancer treatment, particularly 
anti-CD20 therapy, and lower vaccination response (28, 29). Interestingly, 
lymphocytopenia, which has been also related with a poor vaccine response, was also 
associated with progression to critical infection (30). Finally, among patients that 
progressed to critical infection, vaccination does not seem to have protective effect 
against death, contrary to treatment with monoclonal antibodies with in vitro effect 
against SARS-CoV-2 Omicron variants (23, 31). These findings underline the need for 
effective anti-SARS-CoV-2 treatments in these vulnerable cohorts. 
Of additional concern is the widespread use of immunosuppressive medications to treat 
COVID-19 (32). Indeed, the publication of the RECOVERY trial, which showed a significant 
reduction in mortality among patients with COVID-19 who received dexamethasone 
(33), resulted in a paradigm shift in the medical management of COVID-19, whereby 
immunomodulation and not antiviral therapy has become an accepted clinical practice 
standard. Therefore, current guidelines endorse the use of dexamethasone and IL-6 
inhibitor therapy in subgroups of patients with COVID-19 without excluding 
immunocompromised patients (34), even though these patients were generally 
excluded from clinical trials. Moreover, given that immunosuppressed patients are at 
risk for protracted SARS-CoV-2 infection, concerns have been raised about whether 
immunosuppressive therapies may promote chronic infection and worse outcomes, 
including selection and transmission of new SARS-CoV-2 variants (32). Thus, studies 
among immunocompromised hosts should ideally be focused on identifying direct-
acting antivirals that markedly reduce SARS-CoV-2 replication, be they small molecules 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 36
 
 
 
or antibodies. So far, however, patients with immune-system disorders have not been 
sufficiently represented in the clinical trials for COVID-19 antiviral drugs. At the appendix 
“Material” of ACCT-1 trial of remdesivir, immune-depressed patients are only a 7% of 
patients of the 1,062 patients included (32 in the remdesivir arm and 41 in the placebo 
arm) (35). The number is almost the same for patients with neoplasm malignant with 43 
patients included in the remdesivir arm and 37 in the placebo arm representing 8% of 
the study population (35). Throughout the study analysis, patients with immune-system 
disorders were grouped with patients with other more frequent comorbidities such as 
hypertension and obesity and no subgroup analysis was performed (35). A more recent 
trial, published in 2022 (remdesivir plus dexamethasone versus dexamethasone alone 
also did not have a subgroup of immune-system disorders as comorbidities described 
(36). So, although the efficacy of remdesivir has been questioned due to conflicting data 
(35, 37), this RNA polymerase inhibitor and other antivirals such as molnupiravir 
(NCT04405739) (38) should also be studied in clinical trials of immunocompromised 
hosts who may reap the greatest benefit from such interventions. Regarding the use of 
monoclonal antibodies, which are already available for administration to both 
immunocompromised and immunocompetent patients in the United States via an 
emergency use authorization (EUA), they should now be expanded via clinical trials to 
encompass settings beyond those outlined in the EUA (e.g., early hospitalisation, 
supplement oxygen requirement in patients with prolonged replication), with a focus on 
immunocompromised patients who may benefit the most from them. It should be 
pointed out, however, the marked loss of activity of many monoclonal antibodies 
(MAbs) against SARS-CoV-2 Omicron variants that underscores the importance of 
developing MAbs that target conserved regions of spike receptor-binding-domain (39). 
1.2 
INFORMATION ON THE STUDY DRUG 
In the set of preclinical studies, plitidepsin showed strong antiviral activity in in vitro 
models of SARS-CoV-2 infection, also showing a better therapeutic index than other 
drugs, including remdesivir (40, 41). In fact, highly consistent results were obtained, with 
the IC50 of plitidepsin always being in the nanomolar range, regardless of the coronavirus 
species (HCoV 227E, SARS-CoV, and SARS-CoV-2), the infected host cells, or the 
quantifying method used (42). Notably, a similar in vitro antiviral effect was induced by 
plitidepsin against the α, β, δ, μ, and ο variants of SARS-CoV-2, which are known to bear 
several mutations affecting the viral spike protein that facilitates viral entry through its 
interaction with the hACE2 receptor (Table 1) (42, 43).  
 
Table 1 Plitidepsin in vitro activity against different SARS-CoV-2 variants. 
Variant 
Strain 
IC50 (nM) 
CC50 (nM) 
Selectivity Index 
Wild type 
WA1 
0.20 
11.1 
56 


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Alpha 
B.1.1.7 
0.05 
11.1 
222 
Beta 
B.1.351 
0.08 
11.1 
139 
Delta 
B.1.617.2 
0.09 
11.1 
123 
Mu 
B.1.621 
0.27 
11.1 
41 
Omicron 
B.1.1.529 
0.29 
6.3 
22 
IC50: 50% of maximum SARS-CoV-2 inhibitory capacity; CC50: 50% of maximum cytotoxic effect on host cells (HeLa-
ACE2); Selectivity Index: CC50/IC50. 
 
A recent study showed that plitidepsin treatment (at concentrations in nanomolar 
range) of Vero E6 cells infected with SARS-CoV-2 completely abolished the formation of 
double-membrane vesicles (DMVs), organelles that support coronavirus genome 
replication (44). Additionally, neither SARS-CoV-2 nucleocapsid (N) protein nor double-
stranded RNA (dsRNA) were present in plitidepsin-treated infected cells (44). 
Altogether, these results showed that plitidepsin treatment completely blocked the 
assembly of SARS-CoV-2 viral structures in infected cells. 
In addition, White et al (2021) also demonstrated a strong antiviral activity in vivo, 
characterised by a significant reduction in the viral load in lungs, as well as a clear 
reduction in alveolar and peribronchial inflammation (Figure 1) (40).  
 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 38
 
 
 
 
 
 
Remdesivir, n=3; plitidepsin 0.3 mg/kg and vehicle, n=5.          Taken from (White et al 2021). 
*p<0.05, **p<0.01. 
 
Figure 1 Viral load (top) and histopathology score of inflammation (bottom) in 
the lung of K18-hACE2 mouse model, infected with SARS-CoV-2 and treated 
with plitidepsin. 
 
1.2.1 Plitidepsin 
Plitidepsin is a cyclic depsipeptide that was first isolated from a Mediterranean tunicate 
(Aplidium Albicans). Currently plitidepsin is manufactured by synthesis. 
Plitidepsin was approved in Australia in 2018 for the treatment of relapsed/refractory 
multiple myeloma (oncology) and is in development for the treatment of COVID-19. 
1.2.1.1 Name and Chemical Information 
Plitidepsin is synthesised chemically. The structure is summarised below: 
INN Name: 
plitidepsin 
IUPAC Name: 
(-)-(3S,6R,7S,10R,11S,15S,17S,20S,25aS)-11-hydroxy- 
3-(4-
methoxybenzyl)-2,6,17-trimethyl-15-(1-methylethyl)- 7-[[(2R)-4-methyl-


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2-[methyl[[(2S)-1-(2-oxopropanoyl)pyrrolidin- 
2-
yl]carbonyl]amino]pentanoyl]amino]-10-[(1S)-1-methylpropyl]-20-(2-
methylpropyl)tetradecahydro-15H-pyrrolo[2,1-f]-
[1,15,4,7,10,20]dioxatetrazacyclotricosine-1,4,8,13,16,18,21(17H)-
heptone 
Laboratory 
Names: 
aplidin, aplidine, dehydrodidemnin-B, APLD, DDB, PM90001 
Molecular 
Formula: 
C57H87N7O15 
Molecular Mass: 
1110.3 g/mol 
Structural 
Formula: 
 
1.2.1.2 Mechanism of Action   
The target for plitidepsin’s anti-tumour and anti-viral activity is eukaryotic elongation 
factor 1 alpha (eEF1A).  
In eukaryotic cells, FLIM-FRET experiments demonstrated that plitidepsin localises 
sufficiently close to eEF1A to suggest the formation of drug-protein complexes in the 
cytoplasm (45). A separate set of experiments carried out with 14C-plitidepsin and eEF1A 
purified from rabbit muscle showed that plitidepsin binds eEF1A with high affinity and a 
low rate of dissociation (45).  
As viruses depend on host cell proteins for their replication and propagation, it is not 
surprising that they have apparently evolved to utilize eEF1A in their life cycle (46). One 
of the most abundantly produced proteins within eukaryotic cells infected with 
coronaviruses is the nucleocapsid (N) protein, a structural protein that forms complexes 
with genomic RNA, interacts with the viral membrane protein during virion assembly, 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 40
 
 
 
and plays a critical role in enhancing the efficiency of virus transcription and assembly 
(47). Coronavirus N protein was shown to interact directly with eEF1A (48) (49) (50) and 
viral replication was inhibited by eEF1A knockdown (49) or pharmacologic inhibition (50) 
in host cells, suggesting the interaction between eEF1A and coronavirus N protein is 
essential. 
To determine whether the plitidepsin effect on SARS-CoV-2 virus replication may be 
explained by inhibition of eEF1A, a set of in vitro experiments were performed (40). 
Briefly, human embryonic kidney 293T (HEK293T) cells were co-transfected with a 
lentivirus expressing human angiotensin converting enzyme 2 (hACE2) and with 
different plasmids expressing either EF1A wild type (wt) or EF1A-A399V (which contains 
an amino acid mutation that induces resistance to plitidepsin structurally related 
compounds (51)). Each of these transfected HEK293T-hACE2 cell lines was infected with 
SARS-CoV-2 and the effect of plitidepsin treatment assessed. Results showed an 
increase of the IC90 of plitidepsin by a factor of >10 against infected cells transfected 
with EF1A-A399V compared with EF1A-wt. Additionally, EF1A-A339V CRISPR knock-in 
HEK293T-hACE2 cells infected with SARS-CoV-2 was refractory to the SARS-CoV-2 
antiviral activity of plitidepsin by a factor of >12 as compared to the parental cell line. 
These results confirmed that plitidepsin inhibition of EF1A is the molecular basis for 
plitidepsin antiviral effects. 
 
1.2.1.3 Summary from the Clinical Development of Plitidepsin 
Plitidepsin has been used as an experimental therapy in 947 patients with different types of solid 
tumors and haematological malignancies, across 24 clinical trials. The clinical pharmacokinetics 
(PK) of plitidepsin has been well characterised in phase 2/3 studies involving 193 adults with 
multiple myeloma and phase 1/2 studies involving 509 adults with other types of malignancies 
(Table 2). The administered doses of plitidepsin in these studies ranged from 0.13 to 8.0 mg/m2 
as 1-hour, 3-hour, and 24-hour constant rate IV infusions. Please refer to the Investigator 
Brochure (IB) of plitidepsin for an updated and thorough overview of clinical information.  
 
 
 
 
 
 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 41
 
 
 
Table 2 Clinical Studies and Pooled Analysis Supporting the Plitidepsin Clinical Development 
Program 
Study  
Population 
Doses 
(mg/m²) 
Infusion length & 
regimen 
Patients treated/ 
with PK 
Study 
completion 
Phase 1 Studies In Patients With Solid and Haematological Tumours 
APL-A-001a-98 
(52) 
       Solid tumours, NHL 
0.13 to 4.50 
24-h D1, 8, 15 q4wk 
35/34 
September 2002 
APL-A-001b-98 
(53) 
Solid tumours, NHL 
3.00 to 6.00 
3-h D1, 15 q4wk 
27/26 
August 2002 
APL-A-002-98 (54) 
Solid tumours, NHL 
0.13 to 3.60 
1-h D1, 8, 15 q4wk 
49/46 
December 2002 
APL-A-003-98 (55) 
Solid tumours, NHL 
0.20 to 8.00 
24-h D1, 15 q4wk 
67/48 
April 2003 
APL-A-004-98 (56) 
Solid tumours, NHL 
0.08 – 1.50 
1-h D1-5 q3wk 
37/37 
May 2002 
Phase 1 Combination Studies 
APL-A-006-05 (57) 
Solid tumours, lymphoma, 
with carboplatin 
1.80 – 3.00 
1-h D1, 8, 15 q4wk 
20/20 
September 2007 
APL-A-010-08a 
Solid tumours, lymphomas, 
with gemcitabine or sorafenib 1.80 – 3.00 
1-h D1, 8, 15 q4wk 
44/32 
December 2011 
APL-A-011-08 (58) 
Solid tumours, with 
docetaxel or bevacizumab 
2.80 – 4.80 
3-h D1, 15 q4wk 
18/13 
July 2010 
APL-A-012-13 (59) 
MM, with  bortezomib 
4.00 – 5.00 
3-h D1, 15 q4wk 
22/17 
April 2016 
Phase 2 Studies in Patients with Solid Tumours 
APL-B-001-01 
(60) 
Renal cancer, colorectal 
cancer 
5.00 to 7.00 
24-h D1, 15 q4wk 
81/43 
February 2006 
APL-B-002-02c 
(61) 
Medullary thyroid carcinoma 4.25 to 5.00 
3-h D1, 15 q4wk 
16/16 
February 2006 
APL-B-003-02a,c 
Pancreatic cancer 
5.00 
3-h D1, 15 q4wk 
19/- 
March 2006 
APL-B-004-02c (62) 
Non-small cell lung cancer 
5.00 
3-h D1, 15 q4wk 
21/- 
January 2005 
APL-B-005-02c (63) Carcinoma of the urothelium 
5.00 
3-h D1, 15 q4wk 
21/20 
February 2007 
APL-B-006-02c(64) 
Small-cell lung cancer 
2.00 to 3.20 
1-h D1, 8, 15 q4wk 
19/15 
May 2006 
APL-B-007-02c (65) 
Melanoma 
5.00 
3-h D1, 15 q4wk 
37/37 
September 2005 
APL-B-010-02a,c 
Head and neck cancer 
5.00 
3-h D1, 15 q4wk 
10/- 
March 2005 
APL-B-011-02a,c 
Prostate cancer 
5.00 
3-h D1, 15 q4wk 
8/8 
December 2007 
APL-B-016-05c 
(66) 
Melanoma, with or without 
dacarbazine 
1.80 – 3.20 
1-h D1, 8, 15 q4wk 
84/67 
October 2010 
Phase 2 and phase 3 Studies in Patients with Haematological Tumours  
APL-B-013-02c (67) 
NHL 
3.20 
1-h D1, 8, 15 q4wk 
34/24 
June 2010 
APL-B-014-03c (68) MM, with or without DXM 
5.00 
3-h D1, 15 q4wk 
51/45 
June 2008 
APL-B-015-04a 
ALL 
3.20 
1-h D1, 8, 15 q4wk 
17/17 
July 2006 
APL-C-001-09c 
(69) 
MM, with DXM 
5.00 
3-h D1, 15 q4wk 
204/139 
November 2015 
Human Mass Balance Study 
APL-A-013-13 (70) 
Solid tumours 
7.00 mg FD 
3-h D1, 15 q4wk 
6/6 
January 2016 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 42
 
 
 
a Ongoing clinical trials or unpublished results. 
b Patients with PK pooled from phase 1-3 studies listed above. 
c Studies included in the integrated analysis of AEs after the first infusion of treatment (cycle 1/Day 1) 
ALL: acute lymphoblastic leukaemia; D: day;  DXM: dexamethasone; FD: flat dose; h: hour;  m2: square metre; mg: 
milligram; MM: multiple myeloma; NA: not applicable; NHL: non-Hodgkin’s lymphoma; PK: pharmacokinetic; q: 
every; wk: week 
As shown in the above Table 2 (APL-A-004-98 study), the multi-day 60-min IV infusion schedule 
of plitidepsin was initially explored in a phase 1 trial in oncology (56). The Recommended Dose 
for Phase 2 trials was defined as 1.2 mg/m2/day for 5 consecutive days, and was found to be 
safe in seven patients from this study who received it. These patients received an actual median 
daily dose of 2.2 mg (range 1.9 – 2.7 mg/day), corresponding to a total median dose of 11.2 mg 
over a 5-day period (range: 9.6 – 13.7 mg). 
APLICOV-PC was the first trial developed in COVID-19 (42). It explored three doses of plitidepsin, 
delivered daily over 3 consecutive days: 1.5, 2 and 2.5 mg/day. The three doses showed 
acceptable safety for further development in COVID-19 (See IB v5.0, section 5.3). NEPTUNO is 
the phase III trial in hospitalised patients with moderate COVID-19 requiring oxygen 
supplementation. It explores the efficacy and safety of two doses of plitidepsin (1.5 and 2.5 
mg/day) versus a control group. It has recently been closed for accrual and results are not 
available yet. Nevertheless, a futility safety analysis performed when there were at least 30 
patients per arm with 30-days follow-up did not find any alert signal.  
Regarding the infusion time selection, the clinical development of plitidepsin as an anticancer 
compound (summarized in the above Table 2) initially explored three different lengths of 
infusion, 1-h, 3-h and 24-h. APLICOV-PC, is the only study conducted with plitidepsin that has 
used a 90-min IV daily infusion in a total of 45 patients. The rationale for the daily administration 
schedule of APLICOV-PC was discussed with the Spanish Medicines Agency and stemmed from 
a conservative extrapolation from the results of a phase 1 trial performed in cancer patients 
(56). The 60-minute IV infusion schedule was the one selected for the NEPTUNO trial. The 
population PK model that supported the selected doses of plitidepsin in COVID-19 was used to 
compare the simulated plasma PK profiles when plitidepsin is administered as 1 h and 1.5 h 
infusion (see Figure 2). The expected differences in Cmax between both infusion lengths are 
small. Moreover, Cmax with the 2.5 mg dose given as a 1-h infusion is expected to be 
considerably below median Cmax values achieved in more than 300 patients with different 
tumor types (Figure 3), for whom no safety signals of concern were observed after the first 
infusion of plitidepsin (IB section 5.2.2). 
 
Study  
Population 
Doses 
(mg/m²) 
Infusion length & 
regimen 
Patients treated/ 
with PK 
Study 
completion 
Phase 1 Study in Patients with COVID-19 
APLICOV-PC (42) 
COVID-19 
1.5 to 2.5 mg FD 
1.5-h D1-3 
45/- 
Nov 2020 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 43
 
 
 
 
Figure 1 Total plasma concentration profiles vs plitidepsin time predicted between 1 hour and 
1.5 hour.  
 
In NEPTUNO Phase III trial, a futility analysis was performed with at least 30 patients per arm 
and 30-days follow-up. No alert signals were found, as concluded by the IDMC, reinforcing the 
safety of the 60-min IV infusion scheme previously assayed the trials displayed in Table 2. 
The IB tabulates the safety of multiple treatments of plitidepsin delivered weekly at 60-min IV 
infusion, at the recommended safe dose of 3.2 mg/m2 (IB: Table 7 – group C), higher than the 
daily flat dose of 2.5 mg. 
In parallel, the compassionate use of plitidepsin was granted to treat immune-compromised 
patients with COVID-19, not eligible for NEPTUNO trial. This experience has been compiled by 
the treating physicians as a retrospective analysis on 33 patients (71). The majority of the 
patients received the dose of 2.5 mg/day, except for 4 frail patients with organ dysfunction, who 
received 1.5 mg. A total of 111 doses of plitidepsin have been delivered as 60-min IV infusions, 
with a lack of infusion-related and serious systemic adverse events. With the caveat of being a 
retrospective study, this information is important as mimics the target population expected to 
be included into NEREIDA trial. 
 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 44
 
 
 
 
Figure 2 Visual predictive check describing the time course of plitidepsin plasma in different 
tumor types. Blue lines and areas represent the median and 95% confidence intervals of the 5, 
50 and 95th percentiles of the model-based predictions, and dots represents the plasma 
plitidepsin concentrations. 
 
 
1.2.1.3.1 Benefit-Risk Considerations in COVID-19 
Plitidepsin has also been evaluated in a proof of concept (POC) study (APLICOV-PC) that included 
46 hospitalised COVID-19 patients enrolled across 10 sites in Spain. As mentioned above, three 
doses of plitidepsin delivered as a 90-minute IV infusion were studied in this trial: 1.5, 2, and 2.5 
mg, daily for 3 consecutive days (corresponding to 4.5, 6, and 7.5 mg per single course). Safety 
was the primary outcome of the POC study, which was assessed by the frequency of grade ≥ 3 
AEs based on NCI CTCAE v.5.0 criteria, the percentage of patients with treatment-related and 
unrelated AEs, serious adverse events (SAEs), changes from baseline in haematologic and non-
haematologic parameters, and percentages of patients with electrocardiogram (ECG) 
abnormalities (42). 
AEs regardless of relationship are summarised by dose cohort and overall in Table 17 of the IB. 
Overall, 98% (44/45) of patients experienced at least one AE.  The most common AEs by organ 
system, occurring in 2 or more (≥4.4%) of patients overall were: 
□ Haematologic disorders: leukocytosis (6.7%), lymphopenia (6.7%) 
□ Gastrointestinal disorders: abdominal pain (8.9%), constipation (17.8%), diarrhoea (17.8%), 
dyspepsia (8.9%), nausea (42.2%), vomiting (17.8%) 
□ General disorders: asthenia (13.3%), pyrexia (44.4%) 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 45
 
 
 
□ Infections: bacteremia (4.4%), COVID-19 pneumonia (6.7%), pneumonia (4.4%), urinary tract 
infection (4.4%) 
□ Investigations: ALT increased (11.1%), AST increased (6.7%), alkaline phosphatase increased 
(4.4%), C-reactive protein increased (24.4%), CPK increased (4.4%), ferritin increased (31.1%), 
fibrin D-dimer increased (17.8%), GGT increased (13.3%), LDH increased (8.9%), triglycerides 
increased (4.4%) 
□ Metabolic and nutrition disorders: decreased appetite (4.4%), hyperglycaemia (17.8%) 
□ Musculoskeletal disorders: back pain (4.4%) 
□ Nervous system disorders: dizziness (6.7%), dysgeusia (4.4%), headache (13.3%), insomnia 
(8.9%), presyncope (8.9%), somnolence (4.4%) 
□ Psychiatric disorders: delirium (4.4%) 
□ Renal: pre-renal failure (4.4%), renal impairment (4.4%) 
□ Respiratory: acute respiratory distress syndrome (8.9%), acute respiratory failure (4.4%), 
cough (24.4%), dyspnoea (15.6%), hypoxia (4.4%), tachypnea (4.4%) 
□ Vascular disorders: hypertension (4.4%), phlebitis (17.8%) 
 
No significant effects were observed on atrioventricular conduction or on depolarization in the 
protocol-specified analysis of ECGs, as no patient showed  corrected QT interval by Fredericia 
[QTcF] >480 ms or change from baseline in QTcF >60 ms. Additionally, no significant effects were 
observed on atrioventricular conduction or on depolarisation, as measured by mean changes in 
PR and QRS intervals. 
Liver test abnormalities: Although the majority of patients in the APLICOV-PC study experienced 
new or worsening abnormal liver function tests on study (with lab tests graded according to 
CTCAE v5.0), including increased ALT (65.9%), increased AST (29.5%), and increased GGT 
(38.6%), these events were reversible, mild, and short-lasting, as shown in Figure 10 of the IB 
for ALT, and no cases of hepatocellular injury were reported in the APLICOV-PC study. Notably, 
about one-third of patients had elevated liver function tests at baseline, including elevated ALT 
(grade 1 in 34.1% of patients), elevated AST (grade 1 in 36.4%), elevated GGT (grade 1 in 36.4%, 
grade 2 in 6.8% and grade 3 in 2.3%) and elevated LDH (grade 1 in 52.3%), but none had elevated 
bilirubin at baseline and no patients reported liver disease comorbidities at baseline. Although 
other studies of hospitalised COVID-19 patients have reported increased abnormal liver tests 
and increased liver injury with the use of the antiviral agents lopinavir, ritonavir and remdesivir 
(72, 73), there was no evidence of plitidepsin-induced liver injury in the APLICOV-PC study. 
Nausea and vomiting: Nausea and vomiting were the most commonly reported AEs.  A protocol 
amendment “AR9” was implemented in August 2020 to modify the prophylactic premedication 
given prior to plitidepsin infusion. Ondansetron 8 mg IV slow infusion was added and route of 
administration of dexamethasone was changed from oral to IV. The implementation of the 
aforementioned protocol amendment was associated with a reduction in the proportion of 
patients with nausea (from 55.5% to 38.9%) and vomiting (from 22.2% to 13.9%) and no new 
hypersensitivity reactions were seen in any of the 36 patients treated after the amendment (108 
infusions) (See Table 18 of the IB). 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 46
 
 
 
 
Treatment was well tolerated, with equivalent safety outcomes in all three dose cohorts (42). 
Only 31% (14/45) of the patients experienced Grade ≥3 AEs  and only 2 patients experienced a 
treatment-related Grade ≥3 AE: one case of anaphylactic reaction within 5 minutes of starting 
the first plitidepsin infusion that resolved with pharmacologic treatment but resulted in 
treatment discontinuation and one case of diarrhoea that had no impact on study treatment 
(42). 
Considering the low rate of drug-related Grade ≥3 AEs and the high discharge rate at Day 15 
(82% in the full study population; 100%, 96%, and 53% in patients with FDA mild, moderate, and 
severe disease at baseline, respectively), along with an average 3.25-log10 reduction in baseline 
viral load by Day 15, evidence from the APLICOV-PC study indicated a positive benefit-risk for 
plitidepsin for treatment of patients hospitalised for COVID-19 and justified initiation of the 
randomised, controlled NEPTUNO trial to establish tolerance and efficacy. This study is blinded 
to the Sponsor for analysis, and therefore no clinical information can currently be given. 
 
1.2.1.3.2 Effects of Plitidepsin in Immune System 
An integrated analysis on the safety of the recommended doses for Phase 2-3 clinical trials have 
been done on a total of 419 cancer patients after their first infusion of treatment (cycle 1/Day 
1) (Please refer to the IB of plitidepsin). This analysis also evaluated the potential 
immunosuppressive effects of plitidepsin based on the following parameters: 
□ Incidence of infections: The most significant side effect of immunosuppressive drugs is an 
increased risk of infection. Among the 419 cancer patients, 47 (11.21%) patients experienced 
55 infections after their first infusion of treatment (cycle 1/Day 1), none of which were 
considered by Investigators to be related to study drug. A total of 21 infections in 19 (4.5%) 
patients were reported as SAEs, resulting in prolonged hospitalisation for 18 patients and 
death of one patient as a result of a pneumococcal infection and sepsis. Among SAEs for 
infection, those reported in more than one patient were pneumonia (7 patients), respiratory 
tract infection (3), sepsis (2) and infection (2).   
□ Incidence of lymphopenia and neutropenia: Lymphopenia and neutropenia increase 
susceptibility to infection. Among the 419 cancer patients, there were no reports of 
lymphopenia and reports of neutropenia (grade 3) for only 3 (0.7%) patients after the first 
infusion of treatment (cycle 1/Day 1). The low incidence of neutropenia and absence of 
lymphopenia support Investigator assessments that AEs of infections were unrelated to 
plitidepsin treatment. 
□ Incidence of laboratory abnormalities in lymphocytes and neutrophils: Shift tables for the 
change from baseline to the worst grade on study for lymphocyte count and neutrophil count 
showed new or worsening decreases in lymphocyte count for 15.5% (61/394) of evaluable 
patients and new or worsening decreases in neutrophil count for 9.9% (24/396) of evaluable 
patients.  Most patients (75.9% [299/394]) had grade 0-1 lymphocyte count at baseline and 
only 4 (1.3%) of these patients developed grade 3 lymphocyte count decrease on study. 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 47
 
 
 
Similarly, most patients (85.9% [340/396]) had grade 0-1 neutrophil count at baseline and 
only 1 (0.3%) developed grade 3 neutrophil count decrease on study. The variation of 
lymphocytes counts and neutrophil counts after the first infusion of treatment (cycle 1/Day 
1) is presented in Figure 4 and Figure 5, respectively. 
 
 
Figure 4 Time variation of lymphocyte counts after the first infusion of treatment (cycle 1/Day 
1). 
Each line represents the analytical variation of a single patient. Bold blue line connects the median values 
obtained at bins of 2 consecutive time points. Bold red line represents a LOESS polynomic regression. 
Shaded area plots 95% confidence limits. 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 48
 
 
 
 
Figure 5 Time variation of neutrophil counts after the first infusion of treatment (cycle 1/Day1). 
Each line represents the analytical variation of a single patient. Bold blue line connects the median values 
obtained at bins of 2 consecutive time points. Bold red line represents a LOESS polynomic regression. 
Shaded area plots 95% confidence limits. 
In the same way, the potential immunosuppressive effects of plitidepsin were studied in patients 
with COVID-19 participating in APLICOV-PC, by examining: 
□ Incidence of concomitant infections: The most significant side effect of immunosuppressive 
drugs is increased risk of infection. Among the 45 patients hospitalised for management of 
COVID-19 in the APLICOV-PC study, only 10 AEs for infections other than COVID-19 were 
reported, none of which were considered by Investigators to be related to plitidepsin 
treatment.  
□ Incidence of lymphopenia and neutropenia: Lymphopenia and neutropenia increase 
susceptibility to infection. Among the 45 COVID-19 patients, no AE of neutropenia and only 
3 AEs of lymphopenia were reported, none of which were considered by Investigators to be 
related to plitidepsin treatment. 
    Although several studies have examined the causes of lymphopenia during viral infections, 
the mechanisms underlying lymphopenia in COVID-19 patients is not fully understood. 
Possible underlying causes include redistribution of T cells into infected organs, activation 
induced exhaustion, apoptosis, or pyroptosis (74, 75) 
□ Incidence of laboratory abnormalities in lymphocytes and neutrophils: At baseline, 52.3% of 
patients had reduced absolute lymphocyte count (grade 1 in 47.7% and grade 2 in 4.5%) and 
while on study 31.8% of patients developed new or worsening lymphopenia, with acute onset 
(Figure 6). However, no events of new or worsening lymphopenia were considered by 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 49
 
 
 
Investigators to be related to plitidepsin treatment, as low lymphocyte count (lymphopenia) 
is one of the clinical indicators of the severity of SARS-CoV-2 infection (76, 77).  
At baseline, all patients had normal neutrophil counts and while on study only one patient 
developed grade 1 neutropenia. 
An isolated observation of Grade 3 neutropenia was reported in an asymptomatic outpatient 
on the Day 31 follow-up; this patient was also taking metamizole, and the responsible 
investigator deemed that the event was neither clinically relevant nor related to plitidepsin. 
     Notably, despite the fact that 64.4% of patients received dexamethasone for more than 3 
days, they exhibited a median increase in the absolute number of lymphocytes (Figure 6). 
The lack of a control group prevents conclusive evidence, but this could be a very interesting 
finding given that, in addition to glucocorticoid therapy, SARS-CoV-2 infection can also induce 
an early functional exhaustion of cytotoxic lymphocytes that may be responsible for delaying 
immune responses (78, 79). 
□ Prolonged viral replication of SARS-CoV-2: recent publications report prolonged viral 
replication of SARS-CoV-2 in immunocompromised patients (80-82). As discussed in efficacy 
results, the -3.25 log10 mean reduction from baseline viral load at Day 15 in the APLICOV-PC 
study support the hypothesis that plitidepsin has no immunosuppressive effects, as viral 
replication is shortened, not prolonged with plitidepsin treatment. 
 
 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 50
 
 
 
 
Figure 6 Intra-patient Time Variation in Lymphocyte Count per Dose Cohort and Median 
Trends. 
Source: Appendix 16.1.9 – Figure 15.3.13. 
Each colour represents a patient and the bold dark blue line is the median trend, ULN: upper limit of 
normal 
 
1.2.1.3.3 Clinical 
data 
of 
plitidepsin 
in 
immunocompromised patients with COVID-
19  
Recently published data on the compassionate use of plitidepsin in cancer and 
immunocompromised patients with COVID-19 reinforce the safety of the drug (71, 83, 84).  
The first case report described the use of plitidepsin for management of a cancer patient 
infected with COVID-19 while receiving chemotherapy (83). The investigator noted that patients 
with cancer who also are infected with COVID-19 have a poor prognosis and increased risk of 
all-cause mortality. Additionally, cancer treatments are almost always withheld from patients 
with COVID-19, leading to an increased risk of tumor-related morbidities. In this case, a 75-year 
old male with stage IIIB gastric signet ring cell carcinoma was diagnosed with COVID-19 shortly 
after receiving his first course of FOLFOX-4 chemotherapy. After his COVID-19 diagnosis, he 
received two courses of levofloxacin and dexamethasone over 10 days. After 22 days of 
continued illness, he was hospitalised and treated with dexamethasone combined with 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 51
 
 
 
cyclosporine A without any improvement in his signs of respiratory failure; his lymphopenia 
remained unchanged and he continued to test positive for SARS-CoV-2 by RT-PCR. He was then 
given one full course of plitidepsin (2.5 mg once daily for 3 days). He showed a substantial acute 
reduction in viral load 4 and 7 days after initiating plitidepsin treatment (Figure 7). The patient 
was discharged 18 days after plitidepsin treatment, having received a full second course of 
chemotherapy with only a one-week delay from the planned schedule. All three cycles of 
FOLFOX-4 were well tolerated by the patient, with no signs of bone marrow or organ toxicities 
and plitidepsin treatment did not induce any safety signals that would interfere with anticancer 
therapy.  
 
 
Figure 7 Timeline of principle laboratory and microbiological parameters taking Day 1 of 
plitidepsin therapy as reference, and during hospital admission. 
(A) Quantitative viral load (log10 copies/ml) using nasopharyngeal swabs samples. (B) Plasma C-reactive 
protein (mg/l) levels. (C) PAFI, or PaO2/FiO2 ratio in mmHg. (D) Lymphocytes total count (x103 cells mm3) 
during and after plitidepsin therapy. PAFI, a ratio of the partial pressure of oxygen (PaO2) to a fraction of 
inspired oxygen (FiO2) (PaO2/FiO2) (83). 
 
The second case report described the use of plitidepsin for management of a cancer patient 
infected with COVID-19 who had previous anti-CD20 monoclonal antibody-mediated B cell 
depletion and chronic lymphocytic leukemia (CLL) (84). In this case, a 75-year old male with a 
previous history of CLL was diagnosed with COVID-19 after a week of dry cough and tiredness. 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 52
 
 
 
After 22 days of continued illness, he was hospitalised for persistent fever, fatigue, the 
appearance of respiratory insufficiency and ongoing bilateral opacities by chest X-ray; the 
patient had undetectable levels of antibodies against SARS-CoV-2 and had undetectable levels 
of CD19+ and CD20+ B cells in peripheral blood (due to his prior treatment for CLL). The patient 
experienced further clinical and image severity progression and a course of plitidepsin (2.5 mg 
once daily for three days) was initiated 48 days after onset of COVID-19. Clinical improvement 
was apparent shortly after plitidepsin treatment, which led to withdrawal of oxygen therapy 7 
days after the first dose of plitidepsin. On Day 61 after onset of COVID-19, the patient was given 
an IV infusion of human immunoglobulin, followed 24 hours later by a second course of 
plitidepsin.  On Day 74 the patient received his first negative RT-PCR test and was subsequently 
negative for SARS-CoV-2 in three consecutive samples (Figure 8). The patient was discharged 
from hospital on Day 82 and did not experience any signs of COVID-19 relapse during six months 
follow-up. 
 
 
Figure 8 Timeline of main laboratory and microbiological parameters during and after 
plitidepsin therapy. 
Two timepoints have been taken as reference: the number of days from SARS-CoV-2 symptom onset, and 
the number of days since hospital admission. Plitidepsin was administered on Days 49–51 and 65–67 after 
symptom onset. Parameters shown in the clinical course are as follows: A Quantitative viral load (log10 
copies/ml) using nasopharyngeal swabs samples (grey-dotted line). B SpFI (AU) blue line. C Lymphocyte 
total count (cells/mm3) (red line). AU arbitrary units, CsA cyclosporine A, SpFi ratio of oxygen saturation 
in blood (SpO2)/fraction of inspired oxygen (FiO2) at or below 300 AU (84). 
A small observational retrospective study on immunocompromised patients (n=33) treated with 
at least one full course of plitidepsin under compassionate use for the management of COVID-
19 is also being carried out as an investigator-sponsored study (see also Section 1.2.1.3  


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 53
 
 
 
Summary from  the Clinical Development of Plitidepsin) (71). Most of the patients (n=29) 
received the dose of 2.5 mg/day for three consecutive days, except 4 who had significant 
comorbidities or organ dysfunction (71). Most received only one course of therapy (n=27), 
except 6 patients who received a second cycle (71). Patients were elderly, with a mean (SD) age 
of 67.7 (9.8) years, and 75.8% had received at least one dose of COVID-19 vaccine (71). 
Immunocompromised status resulted from the therapy of haematologic malignancy (70% of 
patients), of solid tumor (27.3%), or of an antineutrophil cytoplasm antibody vasculitis (ANCA, 
3%) (71). Most patients had comorbidities, including high blood pressure (30.3%), chronic lung 
disease (15.2%) diabetes mellitus (9.1%), and heart disease (9.1%) (71). 
At onset of plitidepsin treatment, 37.9% of patients had severe pneumonia and 62.1% had 
moderate according to FDA classification; 48.5% were receiving oxygen supplementation; in 
addition to plitidepsin, 6 patients received IV immunoglobulins and 12 received sotrovimab 500 
mg (anti-COVID-19 antibody) (71). The median time from initiation of plitidepsin to SpFi ≥ 315 
was 8 (95% CI 7; 19) days, the median time for independence from oxygen therapy was 11 (95% 
CI 10; 26) days, and the median time to first negative qPCR (Ct > 36) was 17 (95% CI 13; 25) days. 
The mortality rate was 16% (95% CI 3; 37.3) (71).    
1.3 
STUDY RATIONALE 
• Immunocompromised patients are at increased risk for severe and fatal coronavirus 
disease 2019 (COVID-19) outcomes. 
• Patients with immune-system disorders have not been sufficiently represented in the 
clinical trials for COVID-19 antiviral drugs. 
• Concerns have been raised about whether the accepted clinical practice standard to treat 
COVID-19 with weak antiviral agents and immunosuppressive therapies may promote 
chronic infection and worse outcomes in immunosuppressed patients (at risk for 
protracted SARS-CoV-2 infection), including selection and transmission of new SARS-CoV-
2 variants. 
• Studies among immunocompromised hosts should ideally be focused on identifying 
direct-acting antivirals that markedly reduce SARS-CoV-2 replication, be they small 
molecules or antibodies. 
• Evidence of potent antiviral activity of plitidepsin observed in vitro and/or in vivo.  
• Plitidepsin anti-viral activity is “agnostic” to the different SARS-CoV-2 variants. 
• There is no signal supporting clinically significant immunosuppressive potential of 
plitidepsin neither in the context of oncology clinicalsafety analysis after the first infusion 
of treatment nor in the clinical COVID-19 development (APLICOV-PC study). 
• The compassionate use of plitidepsin in immunosuppressed patients has shown: 
- feasibility of plitidepsin in this target population of dismal prognosis. 
- good tolerability of the 2.5 mg daily for three consecutive days.  
- a median time to SARS-CoV-2 RT-PCR negativisation of 17 days from plitidepsin Day 1. 
- a median time to oxygen independence of 13 days and, in line with this, significant 
radiological improvement of COVID-19 pneumonia, with exceptional requirement of 
high-flow oxygen intervention or intensive care unit, frequently limited in this patient 
population.  


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 54
 
 
 
2. STUDY OBJECTIVES 
2.1 PRIMARY OBJECTIVE 
- 
To evaluate efficacy of plitidepsin in pre-specified groups of immunocompromised 
patients with symptomatic COVID-19 requiring hospital care vs control in terms of 
mortality. 
2.2 KEY SECONDARY OBJECTIVE 
- 
To compare efficacy of plitidepsin vs the control in terms of viral clearance, in each 
group. 
2.3 SECONDARY AND EXPLORATORY OBJECTIVES 
2.3.1 Efficacy secondary objectives 
 
- 
To compare efficacy of plitidepsin vs the control in terms of sustained end of 
hospital care, in each group. 
- 
To compare efficacy of plitidepsin vs the control in terms of symptomatic 
improvement, in each group. 
- 
To compare efficacy of plitidepsin vs the control in terms of clinical status (11 
category WHO Clinical Progression Scale), in each group. 
- 
To compare efficacy of plitidepsin vs control in terms of the need of any kind of 
supplementary oxygen, in each group. 
 
2.3.2 Safety secondary objectives 
 
- 
To compare safety/tolerability of plitidepsin vs the control in terms of adverse 
events, adverse reactions and mortality, in each group. 
- 
To compare safety/tolerability of plitidepsin vs the control in terms of abnormal 
laboratory parameters, in each group. 
- 
To compare safety/tolerability of plitidepsin vs the control in terms of variations 
of vital signs, in each group. 
 
2.3.3 Other secondary objectives 
 
- 
To compare efficacy of plitidepsin vs the control in the need of any type of 
advanced oxygen support (high-flow nasal oxygen, extracorporeal membrane 
oxygenation (ECMO), or non-invasive or invasive mechanical ventilation), in each 
group. 
- 
To compare efficacy of plitidepsin vs the control in the need of intensification of 
respiratory or intensive care support, in each group. 


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- 
To compare efficacy of plitidepsin vs the control in the need of intensification of 
pharmacological therapies for COVID-19, in each group. 
- 
To compare efficacy of plitidepsin vs control in terms of superinfection, in each 
group. 
- 
To compare efficacy of plitidepsin vs the control in terms of all-cause- and related 
to COVID-19-mortality, in each group. 
- 
To compare efficacy of plitidepsin vs the control in terms of subsequent hospital 
admissions, in each group. 
- 
To compare efficacy of plitidepsin vs the control in terms of the time course of viral 
load, in each group. 
- 
To compare efficacy of plitidepsin vs the control in terms of the evolution of 
inflammatory markers, in each group. 
- 
To compare efficacy of plitidepsin vs the control in terms of the immune response 
against SARS-CoV-2, in each group. 
- 
To compare efficacy of plitidepsin vs the control in terms of radiological evolution, 
in each group. 
- 
To compare efficacy of plitidepsin vs the control in terms of restoration of the 
therapy for the underlying disease, in each group. 
- 
To compare efficacy and safety/tolerability of all pooled plitidepsin arms versus all 
control arms in all the aforementioned endpoints. 
- 
To compare efficacy and safety/tolerability between plitidepsin arms (across 
different groups). 
- 
To explore prognostic/predictive factors for clinical deterioration or mortality or 
drug response. 
- 
To increase pharmacology knowledge of plitidepsin. 
 
3. OVERALL STUDY DESIGN 
This is a multicentre, randomised, controlled, open-label, pragmatic, phase 2 basket study to 
assess the efficacy and safety of plitidepsin in different groups of immunocompromised patients 
with coronavirus disease 2019 (COVID-19) who require hospital care.  
Approximately 150 patients are foreseen for the study, divided in 4 different groups of 
immunocompromised patients: 
• 
Group 1 –Patients receiving immune-suppression due to haematopoietic or organ 
transplantation. 
• 
Group 2 – Patients receiving B-cell depleting therapies. 
• 
Group 3 – Patients receiving other immune-suppressive therapies. 
• 
Group 4* – Other situations with immune deficiencies. 
*Not applicable in Spain 
 
Whenever a patient can be allocated to more than 1 group, the priority will be group 1 > group 
2 > group 3 > group 4*. In groups 1, 2, and 3, patients will be randomised to receive best standard 


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care (BSC) ± other antiviral (if clinically indicated) on control arm and BSC + plitidepsin on 
experimental arm. Group 4* will not be controlled, as shown in Figure 9. 
 
*Not applicable in Spain 
 
 
 
Figure 9 Algorithm for Patient Assignment in NEREIDA Basket Trial. 
 
Stratification:  
• 
Group 1: ISARIC mortality (<9/≥9) | Type of graft (allogeneic haematopoietic/lung or 
intestinal/other) 
• 
Group 2: ISARIC mortality (<9/≥9) | Disease (haematological neoplasm/other) 
• 
Group 3: ISARIC mortality (<9/≥9) | Disease (neoplasia/other) 
• 
Group 4*: Not applicable 
*Not applicable in Spain 
 
*Not applicable in Spain


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Study periods: 
The study will include a Screening period, a Treatment period and a Follow-up period.  
The Screening period starts once the patient has provided written informed consent and ends 
when the patient is randomised in the study. Parameters assessed during this time will serve as 
the baseline values. The screening procedures should be preferably performed within 24 h prior 
to administration of study treatment, allowing a maximum of 48 h prior to administration of 
study treatment if required.  
The Treatment period starts on Day 1 (date of randomisation) to 24 h after the last dose of the 
antiviral therapy; if no antiviral was given in the control arm, then the treatment period will 
finish on Day 4, for data management purposes. Antiviral treatment must always be started on 
the randomisation date.  
The Follow-up period starts after the end-of-treatment and lasts until the end-of-study. 
The End of Study (EOS) is Day 60 (±3) or date of early study termination unless ongoing SAEs, if 
applicable.  
The End of Trial (EOT) will be when the Last Patient Last Visit (LPLV) has occurred. 
All patients will receive the best available care as per applicable local, institutional, national, 
supranational COVID-19 treatment guidelines.  
Patients assigned to the experimental arms will receive, in addition to BSC, plitidepsin as a 60-
min IV infusion, during Days 1 to 3.  
Patients randomised to the control arm may receive a regulatory-approved antiviral treatment 
in addition to the BSC. 
For prevention of infusion-related (i.e., plitidepsin-related) reactions (IRRs), allergic reactions 
and emesis reactions, administration of premedications is mandatory in the experimental arms.  
All consenting patients discontinuing early from the study before the EOS visit should complete 
the early termination/withdrawal procedures. 
Independent Data Monitoring Committee (IDMC) 
An IDMC will be established to provide study oversight considering that this is a multicentre, 
randomised study being performed in a population at high risk for morbidity and mortality. The 
IDMC will be established and operated in compliance with the FDA Guidance for Industry 
“Establishment and Operation of Clinical Trial Data Monitoring Committees”. 
The IDMC will be composed of individuals external not only to the study Sponsor, but also to the  
trial managers (including Sponsor’s and Contract Research Organisation’s [CRO] medical 
monitors), and study investigators, and will be comprised of at least 1 clinician specialised in the 
treatment of COVID-19 patients, 1 clinician specialised in assessment of clinical study safety 
issues, and 1 biostatistician specialised in analysis of clinical trials. One IDMC member will serve 
as chair and will prepare minutes of each IDMC meeting, which will be provided to the study 
Sponsor, Sponsor’s medical monitor, and CRO project manager only at the end of the study.  


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The IDMC will be held before enrolling the first patient on study to discuss the protocol and 
analytic plan, ICF, and plans for IDMC monitoring of study safety and effectiveness data. 
The IDMC will have responsibility for: 
• 
Review of all SAEs. 
• 
Review of safety/efficacy trends, such as an accumulating number of deaths in the 
study (i.e., from administration of the first dose of study drug on Day 1 through 
Day 60), to determine if there is a difference between plitidepsin and control groups 
and potential impact on study conduct. 
• 
Each group will proceed with per protocol pre-established recruitment and follow-
up and the IDMC Support Team will provide the IDMC Chairperson with unblinded 
information on the number of deaths and SAEs. Upon review of this data, if either 
the difference in the percentage of patients with deaths or SAEs Grade >3 is at least 
20% higher in any IMP group in comparison with the Control Group, IDMC 
Chairperson will contact the other members of the IDMC to assess the continuity of 
the other groups. 
The IDMC can ask for a temporary halt of the clinical trial at any time to better analyse any 
potential benefit/risk concern. 
 
Study “Stopping rules” 
The Sponsor may suspend or terminate the study if: 
• 
New toxicological or pharmacological findings or safety issues that invalidate the earlier 
positive benefit-risk assessment occur; 
• 
Significant safety concerns, according to the Sponsor’s criteria, arise during the conduct 
of the study; 
• 
The development of plitidepsin is discontinued; 
• 
Any other reason (e.g., best interest of the patients, accrual rate makes the completion 
of the study feasible, etc.) leads to this suspension or termination. 
 
 


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3.1 
PRIMARY ENDPOINT 
• Efficacy primary endpoint 
- 
1-month† all-cause mortality rate. †(Day 30 since randomisation). 
3.2 
KEY SECONDARY ENDPOINT. 
- 
Time to confirmed negativisation in SARS-CoV-2 antigen test or RT-PCR Ct > 30.   
3.3 
SECONDARY ENDPOINTS 
3.3.1 Efficacy secondary endpoint 
 
- 
Time to sustained end of COVID-related hospital care from the time of 
randomisation. Sustained discharge is defined as no subsequent admission within 
30 days of initial end of hospital care [up to day 60 (±3)], related to either COVID-19 
or COVID-19 therapy.   
- 
Time to sustained improvement (defined in Section 7.1.3) and resolution of selected 
COVID signs/symptoms (See Appendix 10 - COVID-19 signs/symptoms checklist). 
- 
Distribution of patients according to their clinical status by the 11-category WHO 
Clinical Progression Scale, at Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 (±3) (See 
Appendix 1 – 11-Point World Health Organization (WHO) Clinical Progression Scale).  
- 
Percentage of patients requiring oxygen therapy on Days 4 (±1), 8 (±1), 15 (±1), 30 
(±2), and 60 (±3). 
- 
Time to sustained discontinuation (i.e., at least 7 days) of oxygen supplementation. 
 
3.3.2 Safety secondary endpoints 
 
Frequency of the following events (all-cause and drug-related): 
- 
Treatment-emergent adverse events (TEAEs);  
- 
TEAEs ≥ grade 3 according to the National Cancer Institute [NCI]-Common 
Terminology Criteria for AEs (CTCAE v.5.0);  
- 
Adverse events of special interest (AESIs);  
- 
Serious adverse events (SAEs);  
- 
Drug related Serious Adverse Events (i.e., SARs); 
- 
Adverse events leading to treatment discontinuation; and  
- 
Deaths (COVID-19-related/all). 
- 
Change respect to baseline* in individual study-defined laboratory parameters (See 
Section 5.7, 5.8 and 5.9 and Appendix 8 - Clinical Laboratory Analyses). 
- 
Change respect to baseline* in individual vital signs (See Section 5.7, 5.8 and 5.9). 
*Baseline = latest test performed between screening and drug administration 
 


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3.3.3 Other secondary/exploratory endpoints 
 
- 
Percentage of patients requiring advanced oxygen support on Days 4 (±1), 8 (±1), 15 
(±1), 30 (±2,) and 60 (±3). 
- 
Time to intensification of respiratory support (WHO >5) (See Appendix 1 – 11-Point 
World Health Organization (WHO) Clinical Progression Scale). 
- 
Total duration of advanced oxygen support. 
- 
Percentage of patients requiring high-flow oxygen on Days 4 (±1), 8 (±1), 15 (±1), 30 
(±2), and 60 (±3).  
- 
Total duration of high-flow oxygen therapy per patient. 
- 
Percentage of patients requiring non-invasive mechanical ventilation on Days 4 (±1), 
8 (±1), 15 (±1), 30 (±2), and 60 (±3). 
- 
Total duration of non-invasive mechanical ventilation per patient. 
- 
Percentage of patients requiring invasive mechanical ventilation or ECMO on Days 
4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 (±3). 
- 
Total duration of invasive mechanical ventilation or ECMO per patient  
- 
Percentage of patients requiring admission to ICU on Days 4 (±1), 8 (±1), 15 (±1), 30 
(±2), and 60 (±3). 
- 
Total duration of intensive care unit (ICU) stay. 
- 
Time to onset of additional (i.e., not present at baseline) immune-modulating drugs. 
- 
Percentage of patients receiving immune-modulating drugs on Days 1, 4 (±1), 8 (±1), 
15 (±1), 30 (±2), and 60 (±3). 
- 
Time to onset of additional (i.e., not present at baseline) antiviral drugs. 
- 
Percentage of patients receiving subsequent antiviral drugs (i.e., not present at 
baseline) on Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 (±3). 
- 
Percentage of patients with a new infection by Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), 
and 60 (±3). 
- 
Cumulative mortality (all-cause and related to COVID-19) by Days 4 (±1), 8 (±1), 15 
(±1), 30 (±2), and 60 (±3). 
- 
Percentage of patients in each study group who require subsequent admission 
within 30 days of initial end of hospital care [up to Day 60(±3)], related to either 
COVID-19 or COVID-19 therapy. 
- 
Change respect to Day 1 in the viral load of SARS-CoV-2, measured by RT-PCR, by 
Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), 45 (±2), and 60 (±3), in each study group. 
- 
Percentage of patients in each study group with undetectable viral load, by Days 4 
(±1), 8 (±1), 15 (±1), 30 (±2), 45 (±2), and 60 (±3). 
- 
Time to either undetectable viral load of SARS-CoV-2 or > 2 logs reduction respect 
to Day 1 
- 
Change respect to baseline* in inflammatory and immunological (C- reactive protein 
[CRP], procalcitonin, lactate dehydrogenase [LDH], ferritin, neutrophil-to-


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lymphocyte ratio,  ALC, D-dimer, and multiplex cytokines assay, by Day 4 (±1), 8 (±1), 
15 (±1) and 30 (±2) and 60 (±3). 
- 
Change respect to baseline* in individual serological assessments against 
SARS- CoV- 2, by Days 30 (±2), and 60 (±3). 
- 
Change respect to baseline* in individual T-cell response against SARS-CoV-2 by 
Days 30 (±2), and 60 (±3).  
- 
Change respect to baseline* to Days 15 (±1), and 30 (±2) in chest X-ray findings 
(Brixia score, centrally assessed) (Appendix 9 – Brixia Score). 
- 
Percentage of patients requiring modification of the therapy (drugs, dose or 
schedule) for the underlying disease. 
- 
Time in which pre-scheduled therapies for the control of the underlying disease 
were not able to be administered. 
- 
Risk for mortality / clinical deterioration according to individual clinical 
characteristics and laboratory observations present at baseline*, scores of validated 
scales (e.g., ISARIC-4C), as well as features related to COVID-19 [including SARS-CoV-
2 variant (if available), vaccination status, duration of the infection, and 
inflammation-related parameters [neutrophil-to-lymphocyte ratio (NLR), protein C 
reactive, interleukine-6 (IL-6) and lymphocytes], the underlying disease, and other 
co-morbidities.  
- 
Limited-sampling pharmacokinetics assessment (See Section 7.6). 
- 
PK-PD analysis. 
 
*Baseline = latest test performed between screening and drug administration 
4. SELECTION OF PATIENTS 
Patients must fulfill all the following inclusion/exclusion criteria to be eligible to participate in 
the study. 
4.1 
 INCLUSION CRITERIA 
To be eligible for inclusion into this study, each patient must fulfil the following inclusion criteria 
within 48 hours prior to randomisation: 
General inclusion criteria: 
1. Signed informed consent obtained prior to initiation of any study-specific procedures 
and study treatment; 
2. Patient aged ≥18 years; 
3. Patient diagnosed COVID-19, with the following characteristics: a) A regulatory 
approved test, collected no more than 3 days prior to study randomisation, with either 
a Ct value ≤30 or a positive antigen test; 
b) Presence of any of the selected signs/symptom listed in Appendix 10 - COVID-19 
signs/symptoms checklist within the last 24 h; 


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4. Patient already admitted or requiring hospital care* for symptomatic COVID-19, for 
which at least one antiviral has failed† or cannot be used‡, after a minimum washout 
period of 24h for small molecules (e.g., remdesivir, molnupiravir, nirmaltrevir/ritonavir) 
and 5 days for antiviral monoclonal antibodies (e.g., tixagevimab + cilgavimab) or 
convalescent plasma. 
* The definition of hospital care is based on the need to use a hospital environment (hospital 
ward, day hospital) for treatment administration and/or clinical monitoring of the patient with 
COVID-19.  
† Failure of a prior antiviral is defined as a documented lack of clinical response, plus evidence of 
persisting positivity for SARS-CoV-2 in an appropriate biological sample, determined by a 
regulatory approved test, collected no more than 3 days prior to study randomisation, with 
either a Ct value ≤ 30 (RT-PCR) or a positive antigen test.  
‡  Contraindication, absence of labelled indication, guidelines, or drug unavailability.   
5. Adequate bone marrow, liver, kidney, and metabolic function, defined by the following 
tests performed at local laboratory: 
o Absolute neutrophil count ≥500/mm3 (0.5 x 109/L); 
o Platelet count ≥ 50 000/mm3 (50 x 109/L); 
o Alanine transaminase (ALT) ≤3 x upper limit of normal (ULN) (≤5 x ULN if pre-
existent liver involvement by the underlying disease); 
o Serum bilirubin ≤1.5 x ULN (or direct bilirubin <1.5 x ULN when total bilirubin is 
above ULN); 
o Estimated glomerular filtration rate ≥30 mL/min [CKD-EPI Creatinine Equation 
(2021)] (85). 
 
6. Females of childbearing potential must have a negative serum or urine pregnancy test 
by local laboratory at screening and must be non-lactating. 
7. Females of child-bearing potential must use highly effective contraceptive methods, 
while on study treatment and for 6 months after last dose of plitidepsin. Fertile males 
with partners of childbearing potential must use effective contraception, while on study 
treatment and for 6 months after last dose of plitidepsin (See Appendix 2 – 
Contraception and pregnancy testing). Patients in the control arm must follow 
contraception methods indicated in the approved product information (summary of 
product characteristics [SmPC] or leaflet). If no information is available in the approved 
product information, patients in the control arm must use highly effective (females of 
child-bearing potential) and effective (fertile males with partners of child-bearing 


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potential) contraception for at least one week after the study completion or the time 
indicated based on the investigator’s discretion.  
Group-specific inclusion criteria: 
• 
Group 1 – Patients receiving, within the last 30 days, immune-suppressive therapy due 
to haematopoietic or organ transplantation. 
o Haematopoietic transplantation. 
o Solid Organ Transplantation: 
▪ 
Lung / intestinal. 
▪ 
Other. 
• 
Group 2 – Patients receiving B-cell depleting therapies within the last 6 months*. 
Includes (but is not limited to): 
o Monoclonal antibodies (mAbs) targeting CD19, CD20, CD38, or CD52 (e.g., 
rituximab, ocrelizumab, ofatumumab, daratumumab, alemtuzumab). 
o B-cell activating factor (BAFF) inhibitors (e.g., belimumab). 
o Bruton's tyrosine kinase (BTK) inhibitors (e.g., evobrutinib, ibrutinib). 
o Chimeric antigen receptor T cell therapy (CAR-T) (e.g., anti-CD19 CAR-T cell). 
*Time restriction is not applicable for CAR-T cell therapy. 
• 
Group 3 – Patients receiving, within the last 30 days, other immune-suppressive 
therapies. 
o Other immunosuppressive therapies not including B-cell depleting agents for 
the treatment of auto-immune disorders. 
o Chemotherapy or targeted therapies with immunosuppressive potential for 
solid tumours or haemotological disorders.  
o Chronic glucocorticoids (i.e., equivalent to prednisone ≥ 20 mg/day for more 
than 1 month). 
• 
Group 4* – Other situations with immunodeficiency. 
o Primary immune deficiencies. 
o Human immunodeficiency virus (HIV) infection, with CD4+ T lymphocyte < 200 
cells/µL in the last month.  
o Radiation therapy within the last 3 months- requires documentation of ALC < 
500 cells/µL. 
o Haematological neoplasia or myelodysplasia not currently receiving any therapy 
o Other situations with a documentation of ALC < 500 cells/µL. 
*Not applicable in Spain 
 
Further information about groups and immunosuppressive therapies can be found in 
Appendix 12 – Definition of NEREIDA Study Groups and lists of immunosuppressive 
treatments. 
 


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4.2 
EXCLUSION CRITERIA 
1. Evidence of critical illness, defined by at least one of the following:  
• 
Respiratory failure defined based on resource utilization requiring at least one of 
the following: endotracheal intubation and mechanical ventilation, ECMO, or clinical 
diagnosis of severe acute respiratory distress syndrome with PaO2*/FiO2 ≤ 100.  
*In case a direct measure of PaO2 has not been obtained, it should be imputed 
according to a referenced formula (Ellis or Rice) (Appendix 5 - Imputation of 
PaO2/FiO2 ratio). For sites located over 1000 m altitude above sea level, PaO2/FiO2 
ratio will be adjusted (Appendix 6- Adjustment of PaO2 from a Site at High Altitude; 
See also Appendix 7 for FiO2 imputations from oxygen flow rates). 
• 
Shock requiring vasopressors. 
• 
Multi-organ dysfunction/failure. 
2. (Criterion eliminated and merged with inclusion criterion #4 based on AEMPS 
recommendations). 
3. Any of the following cardiac conditions or risk factors: 
• 
Cardiac infarction or cardiac surgery episode within the last month;  
• 
History of known congenital QT prolongation; 
• 
Known structural cardiomyopathy with abnormal LVEF (<50%); 
• 
Current clinical evidence of heart failure or acute cardiac ischaemia (New York Heart 
Association (NYHA) class III-IV). 
4. Hypersensitivity to the active ingredient or any of the excipients (mannitol, 
macrogolglycerol hydroxystearate, and ethanol) or contraindication to receive 
dexamethasone, antihistamine H1/H2, or anti-serotoninergic 5HT3 agents.  
5. Females who are pregnant or breast-feeding. 
6. Females and males with partners of childbearing potential (females who are not 
surgically sterile or postmenopausal defined as amenorrhoea for >12 months) who are 
not using at least 1 protocol-specified method of contraception. 
7. Any situation currently requiring increasing needs of immune suppressive agents (e.g. 
acute graft rejection, flare of autoimmune disorder, or cytokine storm syndrome). 
8. Any other clinically significant medical condition (including major surgery within the last 
3 weeks before screening) or laboratory abnormality that, in the opinion of the 
investigator, would jeopardise the safety of the patient or potentially impact on patient 
compliance or the safety/efficacy observations in the study. 
9. Participation in another clinical study involving an investigational drug within 30 days 
prior to screening. 


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5. PLAN OF THE STUDY 
5.1 
PLANNED TRIAL PERIODS (INDIVIDUALLY PER PATIENT) 
Patients will be evaluated at scheduled visits: 
• 
The Screening period: From the day patient has provided written informed consent to 
randomisation. The screening procedures should be preferably performed within 24 h 
prior to administration of study treatment, allowing a maximum of 48 h prior to 
administration of study treatment if required.  
• 
The Treatment period: From Day 1 (date of randomisation) to 24 h after the last dose of 
the antiviral therapy. If no antiviral was given in the control arm, then the treatment 
period will finish on Day 4, for data management purposes. Antiviral treatment must 
always be started on the randomisation date.  
• 
The Follow-up period: From the end-of-treatment until the end-of-study. 
• 
The End of Study (EOS) is Day 60 (±3) or date of early study termination unless ongoing 
SAEs, if applicable.  
 
5.2 
DISCONTINUATIONS 
5.2.1 Screening Failure 
Screen failures are defined as patients who consent to participate in the clinical study but are 
not subsequently randomised in 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. Minimal information includes demography, screen failure details, 
eligibility criteria, and any SAEs related to the study participation. Rescreening is not allowed in 
this study. 
5.2.2 Treatment Discontinuation 
Treatment discontinuation occurs when an enrolled patient ceases to receive the study 
treatment regardless of the circumstances.  
The primary reason for any treatment discontinuation will be recorded on the patient’s 
electronic Case Report Form (e-CRF). 
A subject may stop the study treatment but desirably should continue to the end of study. 
Should a patient decide to prematurely discontinue the study treatment (refuses treatment), all 
efforts will be made to complete and report the observations as thoroughly as possible. He/she 
should be asked if he/she can still be contacted for further information. The outcome of that 
discussion should be documented in the medical records. Whenever possible, patients who 
receive at least 1 dose of plitidepsin and withdraw from the study will be followed for at least 
31 days (±3 days) after administration of the last dose of plitidepsin to follow-up on possible 
open TEAEs at withdrawal or for potential late-onset treatment-related TEAEs. 


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Patients will receive the study treatment(s) while it is considered to be in their best interest. 
Specifically, individual treatment of a given patient will be prematurely ended if: 
• 
Unacceptable toxicity despite allowed/applicable medical prophylaxis or therapy. 
• 
Intercurrent illness of sufficient magnitude to preclude safe continuation of the study 
treatment. 
• 
Patient’s refusal (follow-up has to be continued for those patients who refuse 
treatment, but do not withdraw consent) and/or non-compliance with study 
requirements. 
• 
A major protocol deviation that may affect the risk/benefit ratio for the participating 
patient. 
• 
Investigator’s decision. 
A rapid disease progression per se is not an indication for treatment discontinuation and needs 
additional clinical input. Patients who are withdrawn for any reason must not be re-treated in 
the context of this study at any time.  
All cases of treatment discontinuation must be documented (date and reason) in the eCRF. 
Patients will be informed that they have the right to withdraw from the study at any time, 
without prejudice to their medical care, and are not obliged to state their reasons.  
 
5.2.3 Study Discontinuation  
Study discontinuation occurs when an enrolled patient ceases to participate in the study, 
regardless of the reason. Patients will remain on study until patient consent withdrawal, death, 
or the date of study termination (clinical cutoff) established by the Sponsor. The date and reason 
for study discontinuation will be clearly documented in the medical records of the patient.  
If the patient withdraws consent for disclosure of future information, the Sponsor may retain 
and continue to use any data collected before such a withdrawal of consent. Additionally, 
patients may request destruction of any samples taken and not tested, and the investigator must 
document this in the site study records. Refer to the Schedule of Assessments (Appendix 13) for 
data to be collected at the time of study discontinuation and follow-up and for any further 
evaluations that need to be completed.  
Reasons for study discontinuation can include, but are not limited to: 
• 
Investigator’s decision; 
• 
Patient refusal as reason for discontinuation. 
5.2.4 Lost to follow up. 
A patient will be considered lost to follow-up if he/she repeatedly fails to return for scheduled 
visits on Days 8 (±1), 15 (±1), 30 (±2), and 60 (±3), and is unable to be contacted by the study 
site.  
The following actions must be taken if a patient fails to return to the clinic for a required study 
visit: 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 67
 
 
 
• 
The site must attempt to contact the patient and reschedule the missed visit as soon as 
possible, counsel the patient on the importance of maintaining the assigned visit 
schedule, and ascertain whether or not the patient wishes to and/or should continue in 
the study. 
• 
In cases in which the patient is deemed lost to follow-up, the investigator or designee 
must make every effort to regain contact with the patient (where possible, 3 telephone 
calls and, if necessary, a certified letter to the patient’s last known mailing address or 
local equivalent methods). These contact attempts should be documented in the 
patient’s medical record. 
Should the patient continue to be unreachable, he/she will be considered to have withdrawn 
from the study with a primary reason of lost to follow-up. 
5.3 
PROTOCOL DEVIATIONS 
A protocol deviation is defined as any departure from what is described in the protocol of a 
clinical trial approved by an Independent Ethics Committee (IEC)/Institutional Review Board 
(IRB) and Competent Authorities. Therefore, it applies to deviations related to patient inclusion 
and clinical procedures (e.g., assessments to be conducted or parameters to be determined), 
and also to other procedures described in the protocol that concern the Good Clinical Practice 
(GCP) guidelines or ethical issues (e.g., issues related to obtaining the patients’ Informed 
Consent, data reporting, the responsibilities of the Investigator, etc.). 
Deviations with no effects on the risk/benefit ratio of the clinical trial (such as minimal delays in 
assessments or visits) will be distinguished from those that might have an effect on this 
risk/benefit ratio, such as: 
• 
Deviations that might affect the clinical trial objectives, such as those involving the 
inclusion/exclusion criteria (which could mean that the patient is not eligible for the 
trial) and those having an effect on patient evaluability. 
• 
Deviations that might affect the patient’s well-being and/or safety, such as an incorrect 
dosing of the study treatment due to not following dose adjustment specifications or an 
incorrect preparation of the medication. 
• 
Deviations related to the following of GCP guidelines as described in the protocol and 
regulations in force, such as deviations when obtaining the Informed Consent or not 
following the terms established for reporting SAEs, etc. 
No deviations that may have an effect on the risk/benefit ratio of the clinical trial will be 
authorized. All protocol deviations detected during the study will be appropriately documented, 
and those considered particularly relevant (i.e., those related to ethical issues, to fulfillment of 
GCP guidelines and with an effect on the risk/benefit ratio) will be notified, if applicable, to the 
pertinent IEC/IRB and to the Competent Authorities as established by local regulations. 
5.4 
REPLACEMENT OF PATIENTS 
No patients will be replaced. For each group 1-3, all efficacy analyses will be done on Full Analysis 
Set (FAS) population (See 8.2.1 Full Analysis Set (FAS) population).  


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 68
 
 
 
Therefore, accrual in each group will end when the target number of evaluable patients in the 
FAS population has been reached. 
5.5 
PATIENT SCREENING 
The Screening period starts once the patient has provided written informed consent and ends 
when the patient is randomised/enrolled in the study.  
5.5.1 Screening Assessments and procedures 
Parameters assessed during this time may serve as the baseline values (unless repeated before 
Day 1 treatment). The patient will be screened with the aim to complete screening within 24 h 
prior to administration of study treatment but allowing a maximum of 48 h prior to 
administration of study treatment if required. Upon confirmation of eligibility, the patient will 
be randomised and start treatment on the same day. 
 
 
PROCEDURES / ASSESSMENTS 
Patient Registration 
• 
 ICF signature. 
Demographics 
• 
 Demographic data: gender, year of birth, age and 
race, according to the local regulation. 
Medical and 
immunodeficiency history. 
• 
 Medical History 
• 
 Underlying disease causing immune deficiency. 
• 
 Concomitant therapies.  
• 
SARS-CoV-2 Vaccination status (brand and dates of 
vaccines) 
• 
 Prior medications for COVID-19. 
• 
Scheduled therapies for the underlying disease and 
compliance.  
A detailed history of medications and procedures will be 
documented for each patient at screening.  
Any of the medications ongoing before the first dose of the 
study treatment will be recorded, including start/stop dates, 
indication, dose and frequency.  
Specific focus will be done on medications used to treat 
COVID-19 or the underlying disease, including the theoretical 
schedule for next coming administrations 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 69
 
 
 
 
PROCEDURES / ASSESSMENTS 
Clinical evaluation 
• 
 COVID-19-related signs and symptoms checklist 
(Appendix 10 - COVID-19 signs/symptoms checklist) 
and need for hospitalisation. 
• 
 Complete 
physical 
examination. 
Height 
in 
centimetres (cm) will be measured at screening. Body 
weight will be measured in kilograms (kg). Body mass 
index will be calculated. A full physical examination 
will be performed and should include general 
appearance, skin, neck, eyes, throat, lungs, heart, 
abdomen, back, lymph nodes, extremities, and 
neurological examinations, including Glasgow score 
(See Appendix 11 – Glasgow Coma Scale).  
• 
 Clinical status (11-point WHO Clinical Progression 
Scale) 
• 
 Vital signs will include body temperature (°C), SBP 
(mmHg), DBP (mmHg), pulse rate (bpm) and 
respiratory rate (breaths/min). 
• 
 Lung function tests include partial pressure of oxygen 
(PaO2), fraction of inspired oxygen (FiO2) and oxygen 
saturation (SpO2). PaO2/FiO2 ratio will also be 
measured; either Ellis or Rice formulae can be used to 
estimate PaO2/FiO2 ratio (Appendix 5 - Imputation of 
PaO2/FiO2 ratio). 
Detection of SARS-CoV-2 
• 
 qRT-PCR SARS-CoV-2 or SARS-CoV-2 Rapid Antigen 
Test (RAT) 
Documented diagnosis of SARS-CoV-2 infection, determined 
by a regulatory approved test, collected no more than 3 days 
prior to study randomisation, with either a Ct value ≤30 or a 
positive antigen test.  
SARS-CoV-2 variant will be documented at any time during 
the study, if available information exists.  
Minimum set of laboratory 
tests for screening (at site’s 
laboratory) 
 Haematology: differential white blood cells (WBC) counts 
(including neutrophil and lymphocyte counts), platelet count 
(See Appendix 8 – Clinical Laboratory Analyses). 
 Biochemistry: Liver function test (ALT, total bilirubin (direct 
bilirubin if total is abnormally elevated), calculated CrCL [as 
per  Chronic Kidney Disease Epidemiology Collaboration (CKD-


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 70
 
 
 
 
PROCEDURES / ASSESSMENTS 
EPI)], C-reactive protein (CRP), blood urea nitrogen 
(BUN)/urea (See Appendix 8 – Clinical Laboratory Analyses).   
ECG 
A 12-lead ECG will be performed in triplicate. Cardiac rhythm 
will be identified in ECG intervals of at least 30 seconds of 
duration. PR interval, QT interval (raw and QTcF), heart rate 
and QRS complex, with specialist assessment/judgment of 
further evaluation if appropriate. 
Pregnancy test (if patient is 
a WOCBP) 
A urine or serum pregnancy test will be performed for female 
patients of childbearing potential (i.e., female patients who 
are not postmenopausal or surgically sterile) at screening. 
Eligibility assessment  
Documented review of all Inclusion and Exclusion criteria. 
ISARIC-4C mortality score 
Appendix 4 
Adverse events 
AEs that occurred after signature of the informed consent 
form will be reported. 
 
The patient will be a screening failure if not all inclusion criteria are met or if any exclusion 
criterion is met. A patient should only be treated if eligibility criteria are still met according to 
assessments performed closest to first drug administration. 
5.6 PATIENT RANDOMISATION 
Patients will be sequentially allocated in their respective group, following the algorithm depicted 
in Figure 9.  
Central randomisation will be implemented. In groups 1-3, patients will be assigned to the 
experimental or the control arms at a 1:1 ratio. Stratification will be performed in the 
randomised groups. The ISARIC-4C mortality score will be used as a stratification factor in all 
groups (score <9/≥9). In addition, the type of graft (allogeneic haematopoietic/lung or 
intestinal/other) will be used for Group 1, whereas the underlying disease will be considered for 
Groups 2 and 3. For Group 2, the factor categories will be haematological neoplasm vs other 
disease; the respective categories for Group 3 will be neoplasia vs other. Stratification is not 
applicable in the non-randomised Group 4. 
Day 1 is defined as the day of randomisation. Treatment must start within the same natural day.  
 
 


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EudraCT number: 2022-002489-34 
 
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Protocol. Version 3.0 – 22 March 2023                                                                               Page 71
 
 
 
5.7 EVALUATIONS AND PROCEDURES DURING TREATMENT 
 
 
ASSESSMENT/PROCEDURES 
TIME 
Clinical evaluation 
• 
COVID-19-related 
signs and symptoms 
checklist (Appendix 10 
– COVID-19 
signs/symptoms 
checklist).  
• 
Clinical status (11-
point WHO Clinical 
Progression Scale). 
• 
Vital signs will include 
body temperature 
(°C), SBP (mmHg), DBP 
(mmHg), pulse rate 
(bpm) and respiratory 
rate (breaths/min). 
• 
Targeted physical 
examination. A 
targeted physical 
examination will 
include heart, lungs, 
abdomen, extremities 
plus central nervous 
system (CNS) 
assessment, based on 
the investigator’s 
decision as per clinical 
evolution.  
• 
Lung function tests 
include PaO2, FiO2 and 
SpO2. PaO2/FiO2 ratio 
will also be measured; 
either Ellis or Rice 
formulae can be used 
to estimate PaO2/FiO2 
ratio (Appendix 5 - 
Imputation of 
PaO2/FiO2 ratio). 
Days 1, 2, 3, 4 (±1). When applicable, 
these 
assessments 
should 
be 
performed 
before 
treatment 
administration.  


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Protocol Number: AV-APL-B-002-22 
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Protocol. Version 3.0 – 22 March 2023                                                                               Page 72
 
 
 
 
ASSESSMENT/PROCEDURES 
TIME 
• 
 Concomitant 
therapies. 
• 
 Subsequent 
medications for SARS-
CoV-2 infection. 
• 
 Subsequent 
medications for the 
underlying disease. 
Days 1, 2, 3, 4. 
Laboratory tests* 
Haematology: haematocrit, 
red blood cells (RBC), 
differential WBC counts 
(including neutrophil and 
lymphocyte counts), platelet 
count and haemoglobin (See 
Appendix 8 – Clinical 
Laboratory Analyses). 
 
Days 1, 4 (±1), and whenever clinically 
indicated for an AE evaluation. When 
applicable, these assessments should 
be performed before treatment 
administration. 
 Biochemistry: Liver function 
test [ALT, AP, AST∫, albumin, 
total bilirubin (direct bilirubin 
if total is abnormally 
elevated)], lactate 
dehydrogenase (LDH), glucose 
[fasting], sodium, potassium, 
calcium, BUN/urea, gamma 
glutamyl transferase (GGT), 
creatinine, calculated creatine 
clearance [CKD-EPI], creatine 
phosphokinase (CPK), 
amylase, lipase, pro-
calcitonin, ferritin, and CRP 
(See Appendix 8 – Clinical 
Laboratory Analyses).   
Days 1, 4 (±1), and whenever clinically 
indicated for an AE evaluation. When 
applicable, these assessments should 
be performed before treatment 
administration. 
 Coagulation: D-Dimer (See 
Appendix 8 – Clinical 
Laboratory Analyses). 
Day 1, 4 (±1), and whenever clinically 
indicated for AE evaluation. When 
applicable, these assessments should 
be performed before treatment 
administration. 
Urine analysis (semi-
quantitative elemental tests 
Day 
1 
(previous 
to 
treatment 
administration). Repeat whenever 


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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
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Protocol. Version 3.0 – 22 March 2023                                                                               Page 73
 
 
 
 
ASSESSMENT/PROCEDURES 
TIME 
for specific gravity, blood, pH, 
proteins, bilirubin, glucose, 
ketones, nitrites, and 
qualitative analysis of the 
sediment). 
clinically indicated for AE evaluation. 
When applicable, these assessments 
should 
be 
performed 
before 
treatment administration. 
Inflammatory and 
immunological 
biomarkers in blood 
(central laboratory) 
Luminex® Multiplex cytokine 
assessment. 
Days 1, 2, 4 (±1). 
Anti-SARS-CoV-2 
(central laboratory) 
IgG/M anti-protein S and N. 
Day 
1 
previous 
to 
treatment 
administration. 
T-cell response (IFN-) to 
SARS-CoV-2 S-protein 
(Quantiferon®). 
Detection of SARS-
CoV-2 
qRT-PCR in oro/naso-
pharyngeal exudates (central 
laboratory). 
Day 1, 4 (±1). 
Rapid Antigen Test (at site). 
Day 1, 4 (±1). 
ECG** 
Cardiac rhythm will be 
identified in ECG intervals of 
at least 30 seconds of 
duration, PR interval, QT 
interval (raw), heart rate and 
QRS complex, with specialist 
assessment/judgment of 
further evaluation if 
appropriate. 
At screening or Day 1 (previous to 
treatment administration). Repeat on 
Day 4 (±1), and whenever clinically 
indicated for AE evaluation. 
Chest X-ray 
Qualitative evaluation and 
Brixia score (See Appendix 9 – 
Brixia Score). 
(Submission to central review) 
At screening or Day 1 (previous to 
treatment administration). Repeat 
whenever clinically indicated for AE 
evaluation. 
ISARIC-4C 
deterioration score 
Appendix 4 
At screening or Day 1 (previous to 
treatment administration). 
Pharmacokinetics 
(central laboratory) 
(See details in Section 7.6). 
Days 1, 3, 4 (see details in Section 
7.6). 
Antiviral 
Treatment 
administration 
(See details in Section 6.1). 
Days 1-3 in the experimental arm.¥  


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Protocol Number: AV-APL-B-002-22 
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Protocol. Version 3.0 – 22 March 2023                                                                               Page 74
 
 
 
 
ASSESSMENT/PROCEDURES 
TIME 
From Day 1 (date of randomisation) 
to the last dose of the antiviral 
therapy in the control arm.¥ 
Survival status 
 
Throughout the treatment period. 
Hospitalisation status 
 
Throughout the treatment period. 
Adverse events 
As per NCI-CTCAE v.5*** 
Throughout the treatment period.  
* Central laboratory for all the tests, except for screening. 
** Triplicate. 
***  The “AE form” will be used to report signs and symptoms and medical history in case of any 
significant change (improvement or worsening) during the study, as well as for events that occur 
after the first drug infusion or any event related to a study procedure within the study period 
(according to ICH guidelines). 
¥Antiviral treatment period in both arms will end 24 h after administration of the last dose of the 
antiviral therapy. If no antiviral was given in the control arm, then the treatment period will finish 
on Day 4. 
∫See Section 7.2.1 Adverse Events of Special Interest (AESIs). 
 
For patients included in the study (all randomised patients) but never treated, only the 
applicable forms at baseline and off-study visit modules of the e-CRF should be completed but 
SAEs occurring during screening period need to be reported (using paper SAE forms). 
All cases of treatment discontinuation must be documented (date and reason) in the electronic 
case report form (eCRF). A subject may stop the study treatment but desirably should continue 
follow-up to the end of study. 
 
5.8 
EVALUATIONS DURING FOLLOW-UP 
 
 
ASSESSMENT/PROCEDURES 
TIME 
Clinical evaluation 
• 
 COVID-19-related 
signs and symptoms 
checklist (Appendix 10 
– COVID-19 
signs/symptoms 
checklist).  
Daily while admitted, 
upon discharge, 
Day 8 ± 1 
Day 15 ± 1 
Day 30 ± 2 Day 45¥ ± 2 and if 
readmission. 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 75
 
 
 
 
ASSESSMENT/PROCEDURES 
TIME 
• 
 Clinical status (11-
point WHO Clinical 
Progression Scale) 
• 
 Vital signs will include 
body temperature 
(°C), SBP (mmHg), 
DBP (mmHg), pulse 
rate (bpm) and 
respiratory rate 
(breaths/min). 
• 
Targeted physical 
examination. A 
targeted physical 
examination will 
include heart, lungs, 
abdomen, extremities 
plus CNS assessment, 
based on the 
investigator’s decision 
as per clinical 
evolution.  
• 
 Lung function tests 
include PaO2, FiO2 and 
SpO2. PaO2/FiO2 ratio 
will also be measured; 
either Ellis or Rice 
formulae can be used 
to estimate PaO2/FiO2 
ratio (Appendix 5 - 
Imputation of 
PaO2/FiO2 ratio). 
 
¥Only 
assessment of 
COVID-19-
related signs and symptoms checklist 
• 
 Concomitant 
therapies. 
• 
 Subsequent 
medications for SARS 
CoV 2 infection. 
• 
 Subsequent 
medications for the 
underlying disease. 
Throughout follow-up period, if 
changes 
Laboratory tests* 
Haematology: haematocrit, 
RBC, differential WBC counts 
Day 8 ± 1 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 76
 
 
 
 
ASSESSMENT/PROCEDURES 
TIME 
(including neutrophil and 
lymphocyte counts), platelet 
count and haemoglobin (See 
Appendix 8 – Clinical 
Laboratory Analyses). 
 
Day 15 ± 1 
Day 30 ± 2  
If clinically indicated (AE), and 
if readmission 
Biochemistry: Liver function 
test [ALT, AP, AST∫, albumin, 
total bilirubin (direct bilirubin 
if total is abnormally 
elevated)], LDH, glucose 
[fasting], sodium, potassium, 
calcium, BUN/urea, GGT, 
creatinine, calculated creatine 
clearance [CKD-EPI], CPK, 
amylase, lipase, pro-
calcitonin, ferritin, and CRP 
(See Appendix 8 – Clinical 
Laboratory Analyses).   
Coagulation: D-Dimer (See 
Appendix 8 – Clinical 
Laboratory Analyses). 
Urine analysis (semi-
quantitative elemental tests 
for specific gravity, blood, pH, 
proteins, bilirubin, glucose, 
ketones, nitrites, and 
qualitative analysis of the  
sediment). 
Repeat whenever clinically indicated 
for AE evaluation. 
Inflammatory and 
immunological 
biomarkers in 
blood (central 
laboratory) 
Luminex® Multiplex cytokine 
assessment. 
Day 8 ± 1 
Day 15 ± 1 
Day 30 ± 2 
 
Anti-SARS-CoV-2 
(central laboratory) 
IgG/M anti-protein S and N. 
Day 30 ± 2 
T-cell response (IFN-) to 
SARS-CoV-2 S-protein 
(Quantiferon®). 


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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 77
 
 
 
 
ASSESSMENT/PROCEDURES 
TIME 
Detection of SARS-
CoV-2 
qRT-PCR in oro/naso-
pharyngeal exudates (central 
laboratory). 
 
Day 8 ± 1 
Day 15 ± 1 
Day 30 ± 2 
Day 45 ± 2 
Rapid Antigen Test (at site). 
Every other day while admitted, 
Day 8 ± 1 
Day 15 ± 1 
Day 30 ± 2 
Day 45 ± 2 
ECG** 
Cardiac rhythm will be 
identified in ECG intervals of 
at least 30 seconds of 
duration, PR interval, QT 
interval (raw), heart rate and 
QRS complex, with specialist 
assessment/judgment of 
further evaluation if 
appropriate. 
Whenever clinically indicated for AE 
evaluation. 
Chest X-ray 
Qualitative evaluation and 
Brixia score (See Appendix 9 – 
Brixia Score). 
(Submission to central review) 
Day 15 ± 1 
Day 30 ± 2 
 
Pharmacokinetics 
(central laboratory) 
(See details in Section 7.6). 
Day 8 (±1) (see details in Section 7.6). 
Survival status 
 
Throughout the treatment period  
Hospitalisation 
status 
 
Throughout the treatment period 
Adverse events 
As per NCI-CTCAE v.5*** 
Throughout the treatment period.  
 
* Central laboratory for all the tests 
** Triplicate. 
***  The “AE form” will be used to report signs and symptoms and medical history in case of 
any significant change (improvement or worsening) during the study, as well as for events 
that occur after the first drug infusion or any event related to a study procedure within the 
study period (according to ICH guidelines). 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 78
 
 
 
  ∫See Section 7.2.1 Adverse Events of Special Interest (AESIs). 
 
5.9 
EVALUATIONS AT THE END OF STUDY 
The following assessments will be performed at Day 60 ± 3 or at the date of early study 
termination: 
 
 
ASSESSMENT/PROCEDURES 
Clinical evaluation 
• 
 COVID-19-related 
signs 
and 
symptoms 
checklist 
(Appendix 
10 
– 
COVID-19 
signs/symptoms checklist). 
• 
 Clinical 
status 
(11-point 
WHO 
Clinical 
Progression Scale). 
• 
 Vital signs will include body temperature (°C), 
SBP (mmHg), DBP (mmHg), pulse rate (bpm) 
and respiratory rate (breaths/min). 
• 
Targeted physical examination. A targeted 
physical examination will include heart, lungs, 
abdomen, extremities plus CNS assessment, 
based on the investigator’s decision as per 
clinical evolution. Body weight will be 
measured in kilograms (kg). 
• 
Lung function tests include PaO2, FiO2 and 
SpO2. PaO2/FiO2 ratio will also be measured; 
either Ellis or Rice formulae can be used to 
estimate PaO2/FiO2 ratio (Appendix 5 - 
Imputation of PaO2/FiO2 ratio). 
• 
 Concomitant therapies. 
• 
 Subsequent medications for SARS-CoV-2 
infection. 
• 
 Subsequent medications for the underlying 
disease. 
Laboratory tests* 
Haematology: haematocrit, RBC, differential WBC 
counts (including neutrophil and lymphocyte counts), 
platelet count and haemoglobin (See Appendix 8 – 
Clinical Laboratory Analyses). 
 
Biochemistry: Liver function test [ALT, AP, AST∫, 
albumin, total bilirubin (direct bilirubin if total is 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 79
 
 
 
 
ASSESSMENT/PROCEDURES 
abnormally elevated)], LDH, glucose [fasting], sodium, 
potassium, calcium, BUN/urea, GGT, creatinine, 
calculated creatine clearance [CKD-EPI], CPK, amylase, 
lipase, pro-calcitonin, ferritin, and CRP (See Appendix 
8 – Clinical Laboratory Analyses).   
Coagulation: D-Dimer (See Appendix 8 – Clinical 
Laboratory Analyses). 
Urine analysis (semi-quantitative elemental tests for 
specific gravity, blood, pH, proteins, bilirubin, glucose, 
ketones, nitrites, and qualitative analysis of the  
sediment). 
Inflammatory and immunological 
biomarkers in blood (central 
laboratory) 
Luminex® Multiplex cytokine assessment. 
Anti-SARS-CoV-2 (central 
laboratory) 
IgG/M anti-protein S and N. 
T-cell response (IFN-) to SARS-CoV-2 S-protein 
(Quantiferon®). 
Detection of SARS-CoV-2 
qRT-PCR in oro/naso-pharyngeal exudates (central 
laboratory). 
 
Rapid Antigen Test (at site, provided by the Sponsor). 
ECG** 
Only if clinically indicated.  
Cardiac rhythm will be identified in ECG intervals of at 
least 30 seconds of duration, PR interval, QT interval 
(raw), heart rate and QRS complex, with specialist 
assessment/judgment 
of 
further 
evaluation 
if 
appropriate. 
Chest X-ray 
Qualitative evaluation and Brixia score (Appendix 9 – 
Brixia Score). 
(Submission to central review) 
Survival status 
 
Hospitalization status 
 
Adverse events 
As per NCI-CTCAE v.5*** 
* Central laboratory for all the test 
** Triplicate. 


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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 – 22 March 2023                                                                               Page 80
 
 
 
***  The “AE form” will be used to report signs and symptoms and medical history in case of any 
significant change (improvement or worsening) during the study, as well as for events that occur 
after the first drug infusion or any event related to a study procedure within the study period 
(according to ICH guidelines). 
  ∫See Section 7.2.1 Adverse Events of Special Interest (AESIs). 
 
 
6. TREATMENTS 
6.1 
DESCRIPTION OF TREATMENTS 
6.1.1 Drug Formulation and Supply 
Plitidepsin 2 mg powder for concentrate for solution for infusion is presented as a sterile, 
preservative free, white to off-white lyophilised powder/cake in a 10 mL, single-dose, Ph. Eur. 
type I clear-glass vial. 
 
 
 
 
 
Before use, the powder/cake is reconstituted with 4 mL of solvent for plitidepsin to give a 
solution containing plitidepsin 0.5 mg/mL. The reconstituted solution is diluted further in either 
sodium chloride 9 mg/mL (0.9%) solution for infusion (normal saline solution, NSS) or glucose 
50 mg/mL (55) solution for infusion (D5W) for IV infusion.  
6.1.2 Storage conditions 
Plitidepsin powder, 2 mg, must be stored at 5oC ± 3oC (2oC to 8oC). Keep the vials in the outer 
carton to protect from light.  
Solvent for plitidepsin must be stored at 5oC ± 3oC (2oC to 8oC).  
6.1.3 Administration of Study Medications 
The IMP may be administered only by the Investigator or by a member of staff specifically 
authorised by the Investigator, as appropriate. 
6.1.4 Treatment Schedule and Dose 
Plitidepsin will be administered as a 60-min (±5 min) IV infusion, every 24 h (±30 min)* for 3 
consecutive days, at a dose of 2.5 mg. 
For prevention of infusion-related (i.e., plitidepsin-related) reactions (IRRs), allergic reactions 
and emesis reactions, administration of premedications is mandatory in the experimental arms. 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
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*Treatment windows do not apply for the first 30 patients enrolled in this study (experimental 
arm) who will be included in the pharmacokinetics evaluations. 
6.1.5 Prophylactic Medication 
For prevention of plitidepsin related infusion reactions, the following premedications MUST be 
given to each patient and should be completed no more than ~2 hours to 20 minutes before 
initiating each plitidepsin infusion: 
• 
Dexamethasone phosphate* 8 mg IV (equivalent to 6.6 mg of dexamethasone base); 
• 
Dexchlorpheniramine maleate* 5 mg IV (or diphenhydramine hydrochloride 25 mg IV); 
• 
Famotidine* 20 mg IV (or equivalent such as ranitidine 50 mg IV). Oral famotidine is 
acceptable, but in this case the dose should be 40 mg and it must be administered 2 
hours (±15 min) before plitidepsin; 
• 
Palonosetron* 0.25 mg IV (tropisetron 5 mg IV could be considered if palonosetron is 
not available. It should be administered orally (PO)/IV on Days 4 and 5 if tropisetron 5 
mg was administered on Days 1, 2 and 3). 
*Provided by the Sponsor, who is also in charge of the labeling (except when the site uses its 
own medication for the trial). 
6.2 DOSE REDUCTION 
Not applicable 
6.3 CONCOMITANT MEDICATION 
6.3.1 Allowed Medications/Therapies 
Any medications, with the exceptions noted in Section 6.3.2 below, which are considered 
necessary for the patient’s welfare, and which will not interfere with the study medication, may 
be given at the discretion of the Investigator. Medications which MUST be administered as part 
of the study treatment schedule are listed in Section 6.1.5. 
Administration of all concomitant drugs received by the patient during the “on-treatment” 
period of the trial must be reported in the appropriate section of the eCRF along with dosage 
information, dates of administration and reasons for use. Additionally, any unplanned 
diagnostic, therapeutic or surgical procedure performed during the study period must be 
recorded in the eCRF. 
6.3.2 Prohibited Medications/Therapies 
• 
Other investigational agents. 
• 
Concomitant use of plitidepsin and antiviral treatments for COVID-19.  


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• 
Strong CYP3A4 inhibitors and inducers throughout plitidepsin treatment period and 
until 24-h washout period (Appendix 3 - Inhibitors and Inducers of CYP3A4), unless 
medical judgement that they are for the patient’s best interest. 
Investigator may decide to stop the study treatment at any time after the first dose of plitidepsin 
and initiate other antiviral treatments for COVID-19.  
6.3.3 Drug-drug Interactions with Plitidepsin 
There have been no clinical studies on drug interactions. Effect of other medicinal products on 
plitidepsin include: 
Interactions with CYP3A4 inhibitors and inducers: In vitro studies indicate that CYP3A4 is the 
major enzyme involved in plitidepsin metabolism, followed by CYP2A6 and CYP2E1. However, a 
pharmacokinetic population analysis of plitidepsin including 283 patients indicated that 
concomitant administration of CYP3A4 inhibitors and inducers does not affect exposure to 
plitidepsin. Nevertheless, it cannot be ruled out that concomitant treatments that inhibit or 
induce CYP3A4 may modify the efficacy and/or increase the probability of side effects associated 
with plitidepsin, so they should be avoided. 
• 
Co-administration of plitidepsin with strong CYP3A4 inhibitors and inducers should be 
avoided, as they may affect the plasma concentration of plitidepsin, unless medical 
judgement that they are for patient’s best interest. 
• 
Co-administration with moderate CYP3A4 inhibitors and inducers should be used with 
caution, as an effect on plitidepsin exposure cannot be excluded. 
Interactions with CYP3A4 inhibitors: Plitidepsin should not be administered with potent 
inhibitors CYP3A4 (e.g. grapefruit juice, clarithromycin, itraconazole, nefazodone, telithromycin, 
voriconazole). Potent inhibitors CYP3A4 should be discontinued before starting treatment and 
during treatment with plitidepsin. They may be re-administered 24 hours after the last dose of 
plitidepsin. 
Moderate inhibitors CYP3A4 (e.g., aprepitant, diltiazem, erythromycin, fluconazole, verapamil) 
should be used with caution, as increased exposure to plitidepsin cannot be excluded 
Interactions with CYP3A4 inducers: To avoid a decrease in the effectiveness of plitidepsin, 
plitidepsin should not be administrated with potent inducers of CYP3A4 enzyme, such as 
anticonvulsants (phenytoin, phenobarbital or carbamazepine), rifampicin, rifabutin and 
St.John’s wort, unless there are no therapeutic alternatives. The appropriate initial dose for 
patients taking potent inducers has not been defined.  
They may be administered again 24 hours after the last dose of plitidepsin. CYP3A4 moderate 
inducers (e.g., bosetan, modafinil, nafcillin) should be used with caution, as a reduction of 
exposure to plitidepsin cannot be excluded. A list of CYP3A4 inducers and inhibitors is included 
in Appendix 3 - Inhibitors and Inducers of CYP3A4.  
Effect of plitidepsin on other drugs: In vitro studies did not show a potential for plitidepsin to 
inhibit or induce metabolism of other drugs. However, the results for ruling out the potential 
inducing effect of plitidepsin on the CYP2B6 enzyme were inconclusive. The potential effect of 


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plitidepsin on CYP2B6 has not been studied in vivo. Therefore, decreases in plasma 
concentrations of drugs that are substrates of this enzyme cannot be ruled out when co-
administered with plitidepsin. 
The use of any herbal/natural products or other "folk remedies" should be discouraged, but use 
of those products, as well as the use of vitamins, nutritional supplements, and all other 
concomitant medications must be recorded in the eCRF. 
 
6.4 
DRUG ACCOUNTABILITY 
Proper drug accountability will be done by the appropriate trained study personnel. Each study 
site will keep records to allow a comparison of quantities of drug received and used at each site 
for monitoring purposes. The Investigator at each study site will be the person ultimately 
responsible for drug accountability at the site.    
All unused drug supplied by the Sponsor will be properly destroyed at the study site. 
Documentation of this procedure must be provided to the clinical trial monitor. If the Sponsor 
agrees, unused drug supplies may be returned to the drug repository. 
6.5 
TREATMENT COMPLIANCE 
The Investigator is ultimately responsible for supervising compliance with the instructions 
described in this study protocol.  
7. STUDY EVALUATIONS 
7.1 
EFFICACY 
7.1.1 Test for COVID-19 
A SARS-CoV-2 Rapid Antigen Test, provided by the Sponsor, will also be performed at the site by 
the research team on Day 4 until discharge, and always at Days 1, 4 (±1 day), 8 (±1 day), 15 (±1 
day), 30 (±2 days), 45 (±2 days), and 60 (±3 days); if RAT is positive at discharge, RAT should also 
be monitored on a weekly basis until negative. A negative result should be confirmed with a 
second RAT within 48 h, by the investigator team. 
Quantitative PCR to assess viral load on Day 1 before initiation of treatment and on Days 4 (±1 
day), 8 (±1 day), 15 (±1 day), 30 (±2 days), 45 (±2 days), and 60 (±3 days) (see Appendix 13 -
Schedule of Assessments and Procedures), will be performed by a central laboratory. Based on 
these assessments, changes in viral load respect to Day 1 will be calculated for each patient. The 
proportion of patients with either > 2 log 10 reductions or undetectable SARS-CoV-2 on each 
time point will also be assessed.  
Details regarding the collection, processing, storage, and shipment of oro-nasopharyngeal 
samples for qPCR testing will be provided to each investigational site before initiation of the 
study in the form of a laboratory manual. All oro-nasopharyngeal samples for qPCR must be 


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processed and shipped as specified in the laboratory manual to maintain sample integrity. Any 
deviations from the processing steps (e.g., sample collection and processing steps, interim 
storage, or shipping conditions), including any actions taken, must be documented and reported 
to the Sponsor. On a case-by-case basis, the Sponsor may make a determination as to whether 
sample integrity has been compromised. Any deviation from the specified sample handling 
procedure that results in compromised sample integrity will be considered a protocol deviation. 
Samples will be analysed using validated analytical methods, as approved by the Sponsor. 
7.1.2 Chest Imaging 
Chest radiograph abnormalities will be detected by chest X-ray on Day 1, 15 (±1 day), and 30 (±2 
days) and submitted to central review. Chest imaging performed within 48 hours of Day 1 is 
accepted and does not have to be repeated. Images will be reviewed for the presence of 
pulmonary infiltrates, pleural fluid, atelectasis, pulmonary oedema, and other findings and 
scored using Brixia criteria (86). 
7.1.3 Clinical Status 
Clinical status (including the need for oxygen supplementation) will be assessed at least once a 
day, or whenever it changes, while receiving hospital care and on Days 4 (±1 day), 8 (±1 day), 15 
(±1 day), 30 (±2 days), 45 (±2 days) and 60 (±3 days), using the 11-point WHO Clinical Progression 
Scale (Appendix 1 – 11-Point World Health Organization (WHO) Clinical Progression Scale). 
COVID-19 signs or symptoms will be systematically collected and scored according to a check list 
(Appendix 10 - COVID-19 signs/symptoms checklist) together with the WHO Clinical Status. 
Research team will record a daily severity rating of their symptom severity over the past 24 hours 
based on NCI-CTCAE v5.0 criteria. Sense of smell and sense of taste will each be rated on a 3-
point Likert scale where: “0” is reported if the sense of smell/taste was the same as usual, “1” if 
the sense of smell/taste was less than usual, and “2” for no sense of smell/taste. 
Sustained improvement of all targeted COVID-19 signs/symptoms is defined as the event 
occurring on the first of 4 consecutive days when all symptoms scored as NCI-CTCAE category of 
moderate-severe intensity, or requiring medical intervention, or limiting instrumental ADL (see 
Appendix 10 – COVID-19 signs/symptoms checklist) are scored as mild or absent AND all 
symptoms scored mild or 0 (absent) at study entry are scored as 0.  
The first day of the 4 consecutive-day period will be considered the First Event Date. 
Sustained resolution is defined as when all targeted symptoms are scored as 0 for 4 consecutive 
days. The first day of the 4 consecutive-day period will be considered the First Event Date. 
For any symptoms not present at baseline, it must be absent at Day 30 (+/-2) to be counted as 
sustained alleviation/resolution.  
7.1.4 Other Efficacy Assessments 
The study day (from Day 1 to Day 60 (±3 days)) on which the following events occurred will be 
recorded: 


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• 
Initiation of supplemental oxygen (of any type) 
• 
Discontinuation of supplemental oxygen (of any type) 
• 
Initiation of respiratory support 
• 
Discontinuation of respiratory support 
• 
Initiation of antivirals or immune-modulating drugs 
• 
Discontinuation of antivirals or immune-modulating drugs 
• 
ICU admission 
• 
ICU discharge 
• 
Subsequent hospital admission for COVID-19 signs or symptoms 
• 
Hospital discharge 
• 
Death 
Additionally, the following information will be gathered for each patient on Days 4, 8 (±1 day), 
15 (±1 day), and 30 (±2 days) (yes/no): 
• 
Receiving supplementary oxygen therapy 
• 
Receiving high-flow oxygen 
• 
Receiving non-invasive mechanical ventilation 
• 
Receiving invasive mechanical ventilation or ECMO 
• 
Requiring hospital admission related to COVID-19 
• 
Requiring ICU admission 
• 
Receiving subsequent antiviral therapies or immunomodulatory  
• 
Occurrence of new infection 
• 
Death 
Based on these data, secondary endpoints for percentages of patients requiring oxygen therapy 
or different types of ventilation will be calculated and the time (in days) to sustained hospital 
discharge or withdrawal of supplemental oxygen therapy, percentages of patients with new 
infection or receiving subsequent antiviral or immunomodulatory therapies, hospitalisation in 
ICU, and hospitalisation will be measured.  
Laboratory based efficacy assessments that are to be performed during the study include: 
• 
Proinflamatory biomarkers: (CRP, LDH, ferritin, cytokine multiplex Luminex® panel, 
including IL-1β, IL-6, IL-10, and TNFα) in each study arm from baseline (Day 1 prior to 
start of the study treatment) to on Days 4 (±1 day), 8 (±1 day), 15 (±1 day), 30 (±2 days), 
and 60 (±3 days),  
• 
Anti-SARS-CoV-2 serologic and cellular response will be centrally assessed by validated 
tests, on Days 1, 30 (±2 days), and and 60 (±3). 
7.2 
SAFETY AND TOLERABILITY ASSESSMENTS 
Safety will be assessed throughout the study through clinical and laboratory safety evaluations. 
These include medical history at screening, physical examinations, vital sign measurements, 
saturation of oxygen (SpO2) by pulse oximetry (or arterial blood gas analyses [PaO2]), and its 
respective FiO2 (Appendix 7 - Imputation of FiO2 from oxygen flow values. High-flow oxygen 


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therapy), triplicate 12-lead ECGs, laboratory tests (haematology, coagulation, and chemistry), 
clinical AE monitoring, and verification of concomitant treatments. 
Patients will be evaluable for safety if they have received any partial or complete treatment 
administration.  
All adverse events (AE) and adverse reactions (ARs), based on clinical signs and symptoms and 
laboratory measurements, will be measured daily until Day 60 (±3 days), except treatment-
related adverse events, treatment-emergent adverse events of special interest, and SAES that 
will be measured daily until resolution or stabilisation to at least Grade 1, or to an acceptable 
level according to the investigator and the Sponsor of his/her designated representative.  
Adverse events will be elicited from the patient (or, when appropriate, from a caregiver, 
surrogate, or the patient’s legally authorised representative) by the study site staff using a non-
leading question such as “How are you feeling today?” or “Have you had any health concerns 
since your last visit?”  
The investigator and any designees are responsible for detecting, documenting, and reporting 
events that meet the definition of an AE or SAE and remain responsible for following up AEs that 
are serious, considered related to the study treatment or the study, or that caused the patient 
to discontinue the study treatment (see Section 7.4.2). 
Clinical Signs and Symptoms: AEs, including worsening of baseline signs and symptoms of 
disease observed by the investigator (preferably by the same physician for a same patient) or 
reported by patients to study nurses will be recorded and graded according to NCI-CTCAE 
version 
5.0, 
accessible 
via 
internet 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_Quick
_Reference_8.5x11.pdf). All events, including those considered not related to the study drugs, 
must be reported in the eCRF. 
Laboratory Abnormalities: Laboratory abnormalities will be recorded and graded according to 
NCI-CTCAE 
version 
5.0, 
accessible 
via 
internet 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_Quick
_Reference_8.5x11.pdf). Laboratory abnormalities that are of clinical significance will be 
recorded in Adverse Event eCRF. 
Laboratory abnormalities for which NCI CTCAE grading is not available will be classified as the 
fold below the lower limit of normal (<LLN) or fold above the ULN. 
All AEs will be graded according to the NCI-CTCAE v.5. Treatment modifications and reason for 
treatment discontinuation will be monitored throughout the study. The latest MedDRA 
dictionary version will be used for AE coding.  
The safety profile of patients will be monitored throughout the treatment and at Day 60 (±3 
days), or until the date of death, whichever occurs first. 
Any treatment-related AEs, treatment-emergent adverse events of special interest, and SAES 
will be measured daily until resolution or stabilisation to at least Grade 1, or to an acceptable 
level according to the investigator and the Sponsor of his/her designated representative. 


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7.2.1 Adverse Event of Special Interest 
The following AEs will be considered and monitored as AEs of special interest (AESIs): 
musculoskeletal disorders, CPK increases, and rhabdomyolysis; hypersensitivity reactions, 
cardiac events and transaminase elevations, thromboembolic events, hepatobiliary disorders, 
and healthcare-associated infections (87).  
If at any time AST/ALT levels are greater than 3 x ULN (> 5 x ULN if pre-existent liver involvement 
at baseline by the underlying disease, with baseline ALT levels between 3-5 x ULN): 
• 
Report as an AESI. 
• 
The investigator should ensure that serum liver enzyme tests, serum bilirubin, and 
coagulation tests (INR or equivalent) are repeated two or three times weekly (local labs). 
Frequency of re-testing can decrease to once a week or less if abnormalities stabilise or 
the trial drug has been discontinued and the subject is asymptomatic.  
• 
The list of concomitant medications should be re-checked, and include nonprescription 
drugs, herbal and supplementary preparations, alcohol and recreational substances.  
• 
Other reasons for liver injury should be ruled out, including co-infection with other virus, 
autoimmune or alcoholic hepatitis, non-alcoholic liver steato-hepatitis, biliary tract 
disease, progression of an underlying disease (e.g. liver metastasis), hypoxic/ischemic 
hepathopathy, or exposure to environmental agents.  
• 
The investigator should consider additional tests to evaluate liver function or structure, 
as well as a consultation to specialists in liver diseases. 
Treatment with plitidepsin must be discontinued in case of: 
• 
ALT or AST > 8 x ULN; 
• 
ALT or AST > 3 x ULN and either total bilirubin >2 x ULN or INR >1.5; 
• 
ALT or AST > 3 x ULN with the appearance of fatigue, nausea, vomiting, right upper 
quadrant pain or tenderness, fever, rash, and/or eosinophilia (>5%). 
7.3 
ADVERSE EVENTS DEFINITIONS  
7.3.1 Adverse Event (AE) 
An AE is any untoward medical occurrence in a patient or clinical investigation patient 
administered a pharmaceutical product which does not necessarily have a causal relationship 
with this treatment.  
An AE can therefore be any unfavorable and unintended sign, (e.g., an abnormal laboratory 
finding), or a disease temporally associated with the use of a medicinal product, whether or not 
considered related to the medicinal product. 
Illnesses with onset during the study or exacerbations of pre-existing illnesses, including but not 
limited to clinically significant changes in physical examination findings and abnormal objective 
tests/procedures findings (e.g., X-ray, electrocardiogram [ECG]) should be recorded. The criteria 
for determining whether an abnormal objective test finding should be reported as an AE are as 
follows: 


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• 
The test result is associated with clinically significant symptoms, and/or 
• 
The test result leads to a discontinuation from the clinical trial, significant additional 
concomitant drug treatment or other therapy, and/or 
• 
The test result leads to any of the outcomes included in the definition of a SAE (see 
definition below), and/or 
• 
The test result is considered to be clinically relevant by the Investigator. 
For the purpose of this protocol, disease progression or worsening of the underlying disease 
should not be reported as AEs. This information will be used only for efficacy assessment. 
7.3.2 Serious Adverse Event (SAE) 
A SAE is any adverse experience occurring at any dose that: 
• 
Results in death (is fatal), 
• 
Is life-threatening, 
• 
Requires or prolongs inpatient hospitalisation, 
• 
Results in persistent or significant disability or incapacity, 
• 
Is a congenital anomaly or birth defect,  
• 
Is medically significant, or 
• 
Is any suspected transmission of an infectious agent via a medicinal product.  
Medical and scientific judgment should be exercised in deciding whether expedited reporting is 
appropriate in other situations, such as an important medical event that may not be immediately 
life-threatening or result in hospitalisation but may jeopardise the patient or may require 
intervention to prevent one of the outcomes listed in the above definition. 
7.3.3 Death 
Death as such is the outcome of a SAE and should not be used as the SAE term itself. The cause 
of death should be recorded as the SAE term instead. When available, the autopsy report will 
be provided to the Sponsor. 
7.3.4 Life-threatening Event 
A life-threatening event is defined as any event in which the patient is at risk of death at the 
time of the event; it does not refer to an event which hypothetically might have caused death if 
it were more severe. 
7.3.5 Hospitalisation/Prolongation of Hospitalisation 
Any event requiring hospitalisation (or prolongation of hospitalisation) that occurs or worsens 
during the course of a patient’s participation in a clinical study must be reported as a SAE unless 
exempted from SAE reporting (see Section 7.4.2). Prolongation of hospitalisation is defined as 
any extension of an inpatient hospitalisation beyond the stay anticipated/required for the initial 
admission, as determined by the Investigator or treating physician. 


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Hospitalisations that do not meet criteria for SAE reporting are:  
a. Reasons 
described 
in 
protocol 
(e.g., 
IMP) 
administration, 
protocol-required 
intervention/investigations]. However, events requiring hospitalisations or prolongation of 
hospitalisation as a result of a complication of therapy administration or clinical trial 
procedures will be reported as SAEs. 
b. Hospitalisation or prolonged hospitalisation for technical, practical or social reasons, in 
absence of an AE. 
c. Pre-planned hospitalisations: any pre-planned surgery or procedure must be documented 
in the source documentation. Only if the pre-planned surgery needs to be performed earlier 
due to a worsening of the condition, should this event (worsened condition) be reported as 
a SAE.  
Other situation that MUST NOT be considered as hospitalisations is the following: 
d. Planned treatments at sites not associated to a hospital and generally considered as minor 
surgical procedures (i.e., laser eye surgery, arthroscopy, etc.). 
7.3.6 Unexpected/Unlisted Adverse Event 
An AE is considered unexpected/unlisted when the nature or severity of which is not consistent 
with the applicable reference safety information. The Sponsor will use the reference safety 
information table included in the most updated IB for plitidepsin for the evaluation of 
listedness/expectedness of adverse events. 
7.3.7 Adverse Reactions 
All untoward and unintended responses to an IMP related to any dose administered. This 
definition covers also medication errors and uses outside what is foreseen in the protocol, 
including overdose, lack of efficacy, misuse and abuse of the product. 
7.3.8 Serious Adverse Reaction (SAR) 
A serious adverse reaction (SAR) is defined as any noxious and unintended response to an IMP 
related to any dose administered that result in death, is life-threatening, requires inpatient 
hospitalization or prolongation of existing hospitalization, results in persistent or significant 
disability or incapacity, or is a congenital anomaly or birth defect. In accordance with ICH-E2A, 
the definition of an adverse reaction implies a reasonable possibility of a causal relationship 
between the adverse event and the IMP. An adverse reaction, in contrast to an adverse event, 
is characterised by the fact that a causal relationship between a medicinal product and an 
occurrence is suspected. It could also be related to the administration procedure when the 
procedure is an essential part of the IMP administration. 
7.3.9 Suspected Unexpected Serious Adverse Reaction (SUSAR) 
Adverse reactions due to medicines administered in a clinical trial that are unexpected and 
serious. 


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7.3.10 Adverse Events Related to the Study Drug 
An AE is considered related to a study drug/IMP if the Investigator’s and/or the Sponsor’s 
assessment of causal relationship to the IMP(s) is “Y (yes)” (see Section 7.3.10). The Investigator 
will assess the causal relationship of the IMP(s) to the SAE. The Sponsor will consider related to 
the study drug(s)/IMP(s) those events for which the Investigator and/or the Sponsor assesses 
the causal relationship with the IMP(s) as “Uk (unknown)” when it cannot rule out a role of the 
IMP(s) in the event. 
7.3.11 Expedited Reporting 
The Sponsor is responsible for the appropriate expedited reporting of suspected unexpected 
serious adverse reactions (SUSAR), including special situations reports (i.e. overdose, medication 
error) and events considered of special interest to the Competent Authorities. The Sponsor will 
also report all SAEs, including those that are associated to special situation reports (overdose, 
medication error), which are unlisted/unexpected and related to the trial drug (IMP) to the 
investigators and to the IEC / IRBs according to current legislation unless otherwise required and 
documented by the IEC / IRBs. A SUSAR that meets the seriousness criteria of life-threatening 
and/or results in death must be reported within seven (7) calendar days. A SUSAR that is not 
life-threatening or does not result in death must be submitted to the regulatory authorities 
within fifteen (15) calendar days. In addition to the regulatory authorities, a SUSAR may also 
need to be reported to other recipients including ethics committee and investigators. In the case 
of a SUSAR which was initially considered to be non-fatal or non-life-threatening, but which 
turns out to be fatal or life-threatening, as soon as possible and in any event not later than 7 
days after the Sponsor became aware of the reaction being fatal or life-threatening. 
7.3.12 Assessment of Causal Relationship to the Study Drug 
The Investigator must provide an assessment of the causal relationship of each SAE to the clinical 
trial IMP(s) according to the following criteria: 
• 
Y (yes): there is a reasonable possibility that the IMP(s) caused the SAE [(serious adverse 
reaction (SAR)]. 
• 
N (not): there is no reasonable possibility that the IMP(s) caused the SAE and other 
causes are more probable. 
• 
UK (Unknown): only to be used in special situations where the Investigator has 
insufficient information (i.e., the patient was not seen at his/her centre and no 
information was shared with the investigator) if none of the above can be used. 
7.4 
ADVERSE EVENTS REPORTING PROCEDURES 
7.4.1 Reporting of Adverse Events 
The Sponsor will collect AEs from ICF signature until Day 60 (±3 days) or until the date of death, 
whichever occurs first. All AEs suspected to be related to the study drug/IMP must be followed-
up after the time of therapy discontinuation until the event or its sequelae resolve or stabilize 
at an acceptable level to the Investigator and the Sponsor. 


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All AEs, including medication errors and uses outside what is foreseen in the protocol, must be 
recorded in English using medical terminology in the source document and the e-CRF. Whenever 
possible, the Investigator will record the main diagnosis instead of the signs and symptoms 
normally included in the diagnoses.  
Investigators must assess severity (grade) of the event following the NCI-CTCAE v.5 and assign a 
relationship to each study drug(s)/IMP(s); and pursue and obtain information adequate both to 
determine the outcome and to assess whether it meets the criteria for classification as a SAE 
requiring immediate notification to the Sponsor or its designated representative. The 
Investigator must provide any relevant information as requested by the Sponsor in addition to 
that already provided in the e-CRF. 
Abnormal laboratory tests occurring during the study should only be recorded in the AE section 
of the e-CRF if the disorder: 
• 
Is associated with clinically significant symptoms, and/or 
• 
Leads to a discontinuation from the study, significant additional concomitant drug 
treatment or other therapy, and/or 
• 
Leads to any of the outcomes included in the definition of a SAE. 
• 
The test result is considered to be clinically relevant by the Investigator. 
Otherwise laboratory results should be reported in the corresponding section of the e-CRF (e.g. 
biochemistry, haematology). 
Signs and symptoms: all signs and symptoms present since the diagnosis of SARS-COV-2 
infection will be collected, including those related with the underlying disease or other 
concomitant conditions only if present at the time of ICF signature. A separate focus will be done 
on targeted COVID-19 signs and symptoms present within the last 24 h prior to screening and 
listed in Appendix 10. They will be also monitored separately by the research team and collected 
in a specific form of the CRF.  
The “Adverse event form” will be used to report targeted signs and symptoms and medical 
history in case of any significant change (improvement or worsening) during the study, as well 
as for events that occur after the first drug infusion or any event related to a study procedure 
within the study period (according to ICH guidelines). 
7.4.2 Reporting of Serious Adverse Events 
The Sponsor will collect SAEs from the time of signing of the ICF until Day 60 days (±3 days) or 
until the date of death, whichever occurs first.  Nonetheless, the Sponsor will evaluate any safety 
information that is spontaneously reported by an Investigator beyond the time frame specified 
in the protocol. 
All SAEs (as defined above) that occur after first study treatment administration regardless of 
relationship to the study drug(s)/IMP(s) must be reported immediately, and always within 24 
hours to the Pharma Mar S.A. Pharmacovigilance Department, electronically by completing the 
applicable e-CRF section. Only in situations of electronic system failure or pregnancy exposure 
during the clinical trial, can SAEs be reported using paper on a “SAE form” by fax 
 
, e-mail 
 or Pharmacovigilance Department telephone 
 


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 for out of office hours (Greenwich Meridian Time [GMT]) assistance on SAE 
reporting. In case of electronic system failure or pregnancy exposure during the clinical trial, 
SAEs initially reported by alternative methods (not electronically), must be followed by a 
completed electronic SAE reporting on e-CRF from the investigational staff within one working 
day. 
SAEs occurring during the screening phase (from ICF signature to randomisation) and after off-
study will be reported using a paper “SAE form” that must be forwarded as mentioned above 
always within 24 hours to the Pharma Mar S.A. Pharmacovigilance Department by fax or email. 
All SAEs, Treatment-related adverse events, and treatment-emergent adverse events of special 
interest must be followed until the event or its sequelae resolves or stabilizes to at least grade 
1, or to an acceptable level according to the Investigator and the Sponsor or his/her designated 
representative. 
7.4.3 Reporting Pregnancy Cases Occurred within the Clinical Trial 
National regulations require that clinical trial Sponsors collect information on pregnancies 
occurring during clinical trials, in which exposure to the IMP(s) at any time during pregnancy, via 
either maternal or paternal exposure, is suspected.  
Therefore, pregnancy and suspected pregnancy (including a positive pregnancy test regardless 
of age or disease state) of a female patient occurring within six months from the patient’s last 
plitidepsin administration, or of the female partner of a male patient occurring while the patient 
is on study drugs or within six months after the last plitidepsin administration, are considered 
immediately reportable events. 
The Investigator will report the following events immediately and always within 24 hours from 
first knowledge: 
• 
Any occurrence of a pregnancy where any kind of exposure to the IMP(s) is suspected. 
• 
Possible exposure of a pregnant woman. 
• 
All reports of elevated/questionable or indeterminate beta human chorionic 
gonadotropins (β-hCGs). 
Immediately after detecting a case of suspected pregnancy in a female patient, the decision on 
her continued participation in the clinical trial will be jointly taken by the patient, the 
Investigator and the Sponsor, with the patient’s best interest in mind. A decision to continue the 
pregnancy will require immediate withdrawal from the trial.  
Any pregnancy, suspected pregnancy, or positive pregnancy test must be reported to Pharma 
Mar S.A. Pharmacovigilance immediately using the Pregnancy Report form. 
The Investigator will follow the pregnancy until completion/termination and must notify the 
outcome of the pregnancy to the Pharmacovigilance Department at PharmaMar within 24 hours 
of first knowledge as a follow-up to the initial Pregnancy report. 
For any event during the pregnancy which meets a seriousness criterion (including fetal or 
neonatal death or congenital anomaly) the Investigator will also follow the procedures for 
reporting SAEs (complete and send the SAE form to Pharma Mar S.A. Pharmacovigilance  within 
24 hours of the Investigator’s knowledge of the event).  


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All neonatal deaths that occur within 30 days of birth should be reported, without regard to 
causality, as SAEs. In addition, any infant death at any time thereafter that the Investigator 
suspects is related to the exposure to the study drug(s)/IMP(s) should also be reported to 
Pharma Mar S.A. Pharmacovigilance within 24 hours of the Investigators’ knowledge of the 
event. 
7.5 
ADVERSE EVENTS MONITORING 
Safety review will be performed at Pharma Mar, S.A. once the SAE forms have been received 
and the e-CRFs have been completed by the Investigator.  
Periodic safety review of clinical data will be performed; an IDMC will be established to provide 
study oversight. The IDMC will be established and operated in compliance with the FDA 
Guidance for Industry “Establishment and Operation of Clinical Trial Data Monitoring 
Committees”. The description of the process will be outlined in the IDMC Charter.  
SAEs will be collected, assessed and reported as per the applicable Regulations by the 
Pharmacovigilance Department.  
Non-serious AEs will be checked for accuracy against the e-CRF during monitoring visits by the 
monitor. 
7.6 
PHARMACOKINETICS 
7.6.1 Blood Sampling 
Blood samples will be collected from the first 30 patients enrolled in this study (experimental 
arms), at the time points depicted in Table 3. In the event that any of the first 30 patients does 
not complete all three doses, the first 30 patients who complete all 3 doses will be included. 
Table 3 Samples for pharmacokinetic evaluations. 
Study 
Day 
Sample 
number 
Time point (absolute time from 
the start of first plitidepsin 
infusion) 
Collection window 
1 
1 
0 h 
Before first infusion 
3 
2 
48 h 
Before SOI (-20 to -1 m) 
3 
49 h 
5 m before EOI (±4 m) 
4 
52 h 
3 h after EOI (±30 m) 
5 
55 h 
6 h after EOI (±1 h) 
4 
6 
72 h 
23 h after EOI (±4 h) 
8 
7 
168 h 
119 h after EOI (±24 h) 
Abbreviations: EOI=end of third infusion; h, hour; min, minute; SOI=start of third infusion 


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The infusion rate will be predetermined to ensure that the doses of plitidepsin are infused in 60 
min, at a constant rate. In order to obtain reliable PK information, the infusion rate should not 
be modified once the infusion begins. If a variation in the infusion time eventually occurs, it is 
very important this to be reflected in the e-CRF. The accurate recording of actual dosing and 
sampling times is much more important than the strict adherence to the scheduled times.  
Blood samples will be obtained into a vacutainer tube by using a peripheral catheter placed in a 
vein of the arm opposite to the side used for drug infusion. Even the last sample must never be 
collected from the catheter used for drug infusion. 
A Laboratory Manual will be provided with details on collection and storage of PK samples. 
Please read it carefully before PK sampling. The blood-containing tubes will be stored frozen 
until their shipment to the Central Laboratory for PK Samples (see details in the Study Contacts). 
All the material for PK procedures will be provided by the Sponsor).  
7.6.2 Analytical Procedures 
Blood samples will be analysed to determine concentrations using validated, specific, and 
sensitive liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) methods 
by or under the supervision of the Sponsor. 
7.6.3 Pharmacokinetic Parameters 
Pharmacokinetic analysis will be the responsibility of the Sponsor in accordance with the current 
Clinical Pharmacokinetics guidelines on population pharmacokinetic analyses (88, 89). Clearance 
and volume of distribution will be the primary parameters of interest for the population PK 
analysis. Additional PK parameters will be calculated, if deemed appropriate. 
 
8. STATISTICAL METHODS 
This is an exploratory basket study to adequately approach the heterogeneity of clinical 
conditions included under the concept of immune compromise. 
The study implements a Bayesian methodology. This decision has been adopted after taking into 
account the relative low frequency of the conditions under study, the relative absence of 
prospective data of the outcomes of COVID-19 for the study populations (most of the available 
data are retrospective and belong to the early waves of the pandemic, with different viral 
variants than nowadays), along with the changing medical practices in these settings. Under 
these premises, a Bayesian approach is more appropriate in terms of efficient decision-making 
process, and will provide novel information for future therapeutic development in these clinical 
conditions (90). 
The trial will use a minimally-informative prior of N(0,1) to update the information provided by 
the trial data to produce a posterior distribution. 


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The resultant posterior distribution will serve as a starting point for further trials within this area, 
whether as a key part of any information data packs provided to experts ahead of an elicitation 
meeting, or directly as the informed prior or hypotheses for further trials (91) (92).   
A calibrated Bayesian hierarchical model (CBHM) described by Chu and Yuan (93) will be used to 
analyse the data, allowing data to be analysed by group and results from each group will be used 
to enrich the data of the other groups. The calibration of the model allows strong information 
sharing when the treatment effect is similar among baskets, and shrinks that sharing towards 0 
as the treatment effect becomes more heterogeneous. 
An overall type I error of 5% was used in analysing by means of credible regions the OR against 
a null of OR=1 [log(OR)=0], the case where no discernible difference is induced from 
intervention. 
Simulation was used to perform sensitivity analysis on a range of informed priors against a 
minimally-informative prior to ensure that its use would not impact the results on the data too 
far.  
The statistical methods for this study are described in a detailed statistical analysis plan (SAP). 
8.1 
SAMPLE SIZE CALCULATION 
Our intention is to conduct this study with 4 groups of immunocompromised patients. 
A) Randomised groups (1-3) 
Patients in three of the four groups will be randomly assigned to one of two arms. Each 
randomised group will have a sample size of 40 patients (20 in the experimental arm, 20 in the 
control arm).  
Given the exploratory nature of this phase II study, this sample size has been chosen based on 
operational considerations to allow the feasibility of accrual in this relatively small population 
and also provide preliminary estimates to establish an updated prior distribution of the mortality 
rates for further investigation using Bayesian methods in a potential Phase III study. 
The operating characteristics of the calibrated Bayesian hierarchical model (CBHM) described by 
Chu and Yuan (93) have been investigated by means of the CBHM package from Yuan at MD 
Anderson Cancer Center (94), and with reference to the JAGS program running in R version 4.2.1 
(95) on Microsoft Windows 10. 
Within the CBHM simulations, 5000 Markov-chain Monte Carlo (MCMC) repetitions per scenario 
were simulated with a range of operating characteristics for the null, alternative and true 
mortality rates moving from 5% to 95% in steps of 5% (Table 4). A maximum sample size of 40 
per randomised group was set through the CBHM simulations, which in turn determines 
whether there is a need for the total sample size to be allocated. Within all simulations, the full 
120 patients were used, suggesting that this is the minimum sample size that enables decision-
making. As an example, if the mortality rate in each group is 20% and we target a clinically 
meaningful reduction of 12.5% in the experimental arm (H0=20%, H1=7.5%), and the assumed 
true mortality rate is 7.5%, the simulation demonstrates that a sample size of 40 per basket will 
allow for a posterior probability around 0.9 in each basket (i.e. 90% probability that the 


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experimental arm is better than the control in each basket), ranging from 0.87 (when the data 
from the fastest basket is analysed) to 0.915 (at the end of the accrual of the last basket) (type I 
error rate= 0.05). 
 
 
Table 4 Example of simulated posterior probabilities for sample size definition.   
 
Mortality 
rate – 
control arm 
(null) 
Mortality rate 
– experimental 
arm 
(alternative) 
True 
mortality 
rate 
Sample 
size per 
basket 
Posterior 
probability 
1st basket 
Posterior 
probability 
2nd basket 
Posterior 
probability 
3rd basket 
20% 
7.5% 
7.5% 
30 
0.82 
0.84 
0.865 
20% 
7.5% 
7.5% 
40 
0.87 
0.88 
0.915 
20% 
7.5% 
7.5% 
50 
0.895 
0.905 
0.93 
20% 
7.5% 
7.5% 
60 
0.905 
0.925 
0.97 
20% 
7.5% 
7.5% 
70 
0.945 
0.98 
0.985 
20% 
7.5% 
7.5% 
80 
0.96 
0.985 
0.985 
 
The full output of the simulations performed can be found in the Statistical Analysis Plan. 
 
B) Non-controlled group 4* 
Many different clinical conditions cannot be classified into one of the 3 randomised groups and 
have been pooled into a fourth group of 30 patients. As there is a remarkable intragroup 
heterogeneity, this group will not be controlled and will contribute to the objective of increasing 
safety knowledge of plitidepsin. A Fleming’s phase II procedure with a 1-stage design has been 
used to estimate the sample size needed to rule out that plitidepsin induces drug-related SAEs 
in at least 35% of the patients (null hypothesis), and to confirm that the respective rate is 10 % 
or lower (alternative hypothesis), with a type I error of 0.05 and a power of 90%.  
If at least 25 patients out of a total sample of 30 do not experience a drug-related SAE, the null 
hypothesis will be rejected. 
*Not applicable in Spain 
8.2 
ANALYSIS POPULATIONS 
8.2.1 Full Analysis Set (FAS) Population 
All randomised patients who have taken at least 1 dose of study treatment (plitidepsin or 
control) and have completed follow-up for survival until day 30 (±2). Patients who die before the 
end of follow-up period will also be included in the FAS population. FAS population will be 
analysed according to their randomised treatment arm.  


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8.2.2 Per Protocol Population 
A subgroup of the FAS population that includes all patients who do not have any important 
protocol deviations that would interfere with the collection or interpretation of the efficacy 
data. Important protocol deviations will be defined in the SAP. Per Protocol population will be 
analysed according to their randomised treatment arm.  
8.2.3 As Treated Population 
All patients who received any exposure to study treatment (plitidepsin or control). As Treated 
population will be analysed according to the treatment they actually received.  
8.3 
GENERAL CONSIDERATIONS 
The design of the trial, the Bayesian analysis and the sample size is based on the 30-day (+/- 2 d) 
survival information on the FAS population. The analysis of the primary and other time-to-event 
efficacy endpoints will be performed primarily in the FAS  but also in the “Per Protocol” and “As 
treated” populations. Supportive analyses will be performed on the Per Protocol population. 
Safety endpoints analyses will be based on the “As treated” population. 
 
8.4 
ANALYSIS METHODS 
8.4.1 Primary Endpoint 
The difference between treatment and placebo arms in all-cause mortality at 30 days will be 
examined using a Bayesian comparison of proportions within the calibrated Bayesian 
hierarchical model (CBHM) framework described by Chu and Yuan (93). The use of the CBHM 
allows for flexible information borrowing across all groups, with strong borrowing in the case 
when treatment effect is homogeneous among the groups and less borrowing as the effect 
becomes more heterogeneous.  
 
In this framework the odds ratio is modelled as θi=log(oddsi) with a normal distribution of 
unknown mean (μ) and variance (σ2). The prior distribution used by the framework will be a 
minimally-informative prior using N(0.1). 
 
The variance (σ2) represents the degree of heterogeneity between the patient groups, with 0 
representing the case where there is homogeneity of treatment effect and thus complete 
pooling of the results across patient groups, with adjustment for targeted p1 rates in each group, 
whereas a value closer to infinity would lead to no borrowing across groups. These estimates 
are iteratively updated within the modelling procedure based on the available data. 
As each basket completes, they will be evaluated for efficacy, with information gained being 
used to update the analyses of successive baskets.   
 
Once all patients recruited to a basket have completed the trial, a formal analysis will be 
performed on the patients within the basket. The result will be able to stand on its own for the 


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specific subpopulation, and the information from this analysis will be used to enrich the 
information at analysis of the proceeding baskets. 
 
Results in later baskets will be related to pooled data, however if one basket is not seen to be 
homogeneous to other baskets, the information from that basket can be run without the pooled 
information from other baskets and its information not pulled through into later analyses. 
 
As a sensitivity analysis the Bayesian hierarchical model will be used.  
 
The cut-off for considering further clinical development of plitidepsin in each randomised group 
will be a posterior probability of superiority for the experimental arm equal to or greater than 
65%. 
Sensitivity/supportive analyses for the primary endpoint will be also performed. Frequentist 
approach for odds ratio comparison: unstratified and stratified considering randomisation and 
actual strata values. 
8.4.2 Secondary Endpoints/Other Endpoints 
Frequency tables will be prepared for categorical variables, and continuous variables will be 
described by means of summary tables, which will include the median, mean, standard 
deviation, interquartile range, minimum, and maximum of each variable. 
In addition to Bayesian methods, frequentist methods will be also calculated. Counts and 
percentages, with their corresponding exact confidence intervals, will be calculated for the 
binomial endpoints. Time-to-event variables and their set time estimates will be analysed 
according to the Kaplan-Meier method and Cox regressions. 
Key secondary endpoint, time to confirmed negativisation in SARS-CoV-2 antigen test or RT-PCR 
Ct > 30, will be analysed according to the Kaplan-Meier method and Cox regressions following 
unstratified and stratified test as supportive analyses.  
8.4.3 Safety Analysis 
Safety analyses will be based on the As Treated Population. All safety parameters will be 
summarised and also listed by patient.  
Treatment-emergent Adverse Events: The verbatim terms used in the eCRF by investigators to 
identify TEAEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) 
and will be graded according to NCI CTCAE version 5.0. Treatment-emergent AEs will be 
summarised by MedDRA system organ class and preferred term. For each TEAE, the percentage 
of subjects who experience at least 1 occurrence of the TEAE will be summarised overall and by 
treatment group. The SAEs, AEs of special interest, and deaths will be listed. All TEAEs resulting 
in discontinuation of study treatment will be listed and summarised by system organ 
class/preferred term. 


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AEs, SAEs, deaths, laboratory evaluations, and study drug discontinuations due to AEs will be 
tabulated in a descriptive way. Counts and percentages will be used for categorical variables, 
and summary tables will be used for continuous variables.  
Any treatment-related adverse events, treatment-emergent adverse events of special interest, 
and SAES that will measured daily until resolution or stabilisation to at least Grade 1, or to an 
acceptable level according to the investigator and the sponsor of his/her designated 
representative. 
Clinical Laboratory Tests: Laboratory data will be summarised by type of laboratory test. The 
worst toxicity grade will be tabulated. Parameters with predefined NCI CTCAE version 5.0 toxicity 
grades will be summarised. Change from baseline to the worst TEAE grade experienced by the 
subject during the study will be provided as shift tables. 
8.4.4 Pooled and Subgroup Analyses 
Pooled and subgroup analyses will be performed, taking into consideration baseline 
characteristics of the patients and of the underlying disease, risks factors for clinical 
deterioration or death, vaccination status, duration of the infection prior to study entry, and 
concomitant therapies (e.g., immunomodulatory agents —including glucocorticoids—, oxygen 
supplementation). 
Subgroup analysis of TEAEs and clinically relevant laboratory tests will be performed based on 
prognostic factors of medical interest. 
8.5 SUMMARY OF ESTIMANDS AND STATISTICAL TESTS 
The primary estimand corresponding to the primary endpoint is defined as: 
Treatment: Participants will be randomised to receive plitidepsin versus control in each 
group. 
Population:  Participants as defined through the inclusion and exclusion criteria, who 
have been randomised to the trial and who have taken at least 1 dose of study 
treatment (plitidepsin or control). 
Variable: 
–1-month all-cause mortality rate (Day 30 since randomisation). 
Intercurrent events: 
–The participant’s data are collected and included in the analysis without regard 
for treatment discontinuation (treatment-policy strategy). 
–In the event of the participant initiating another non-protocol therapy, 1-
month all-cause mortality rate will be evaluated regardless of initiation of new 
non-protocol therapy (treatment-policy strategy).  
Population level summary:  the posterior probability associated for the odds ratio of 1-month 
all-cause mortality rate from a Bayesian logistic regression for each group comparison. 


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Below a summary table where main/supportive analyses are described. P values except for the 
primary analysis have to be considered as exploratory and nominal values will be shown. 
 
Endpoints 
Analyses 
Efficacy primary endpoint 
1-month all-cause mortality rate.  
-Bayesian odds ratio (primary analysis by 
CHBM) 
-Bayesian odds ratio (Berry’s Method) 
- Unstratified odds ratio (Univariate Logistic 
regression) 
- Stratified odds ratio (Multivariate Logistic 
regression) 
 
Key secondary endpoint 
Time to confirmed negativisation in SARS-
CoV-2 antigen test or RT-PCR Ct > 30. 
-Kaplan-Meier (unstratified and stratified)  
-Cox regression (unstratified and stratified) 
Efficacy secondary endpoints 
Time to sustained end of COVID-related 
hospital care from the time of randomisation. 
Sustained discharge is defined as no 
subsequent admission within 30 days of 
initial end of hospital care [up to day 60 (±3)], 
related to either COVID-19 or COVID-19 
therapy 
-Kaplan-Meier (unstratified)  
-Cox regression (unstratified) 
Time to sustained improvement (defined in 
Section 7.1.3) and resolution of selected 
COVID signs/symptoms (See Appendix 10 - 
COVID-19 signs/symptoms checklist). 
-Kaplan-Meier (unstratified)  
-Cox regression (unstratified) 
Distribution of patients according to their 
clinical status by the 11-category WHO 
Clinical Progression Scale, at Days 4 (±1), 8 
(±1), 15 (±1), 30 (±2), and 60 (±3) (See 
Appendix 
1 – 
11-Point 
World 
Health 
Organization (WHO) Clinical Progression 
Scale). 
-Frequencies 
-Unstratified odds ratio (ordinal) 


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Protocol. Version 3.0 – 22 March 2023                                                                               Page 101
 
 
 
Endpoints 
Analyses 
Percentage of patients requiring oxygen 
therapy on Days 4 (±1), 8 (±1), 15 (±1), 30 
(±2), and 60 (±3). 
-Frequencies 
-Unstratified odds ratio (ordinal)  
Time to sustained discontinuation (i.e., at 
least 7 days) of oxygen supplementation. 
-Kaplan-Meier (unstratified)  
-Cox regression (unstratified) 
 
Safety secondary endpoints 
Treatment-emergent adverse events (TEAEs). -Frequencies (severity grade by NCI-CTCAE 
v5) 
TEAEs ≥ grade 3 according to the National 
Cancer Institute [NCI]-Common Terminology 
Criteria for AEs (CTCAE v.5.0). 
-Frequencies 
Adverse events of special interest (AESIs). 
-Frequencies (severity grade by NCI-CTCAE 
v5) 
Serious adverse events (SAEs). 
-Frequencies (severity grade by NCI-CTCAE 
v5) 
Drug related Serious Adverse Events (i.e., 
SARs). 
-Frequencies (severity grade by NCI-CTCAE 
v5) 
Adverse 
events 
leading 
to 
treatment 
discontinuation. 
-Frequencies (severity grade by NCI-CTCAE 
v5) 
Deaths (COVID-19-related/all). 
-Frequencies 
Change respect to baseline in individual 
study-defined laboratory parameters (See 
Section 5.7, 5.8 and 5.9 and Appendix 8 - 
Clinical Laboratory Analyses). 
-Frequencies (severity grade by NCI-CTCAE 
v5) 
Change respect to baseline in individual vital 
signs (See Section 5.7, 5.8 and 5.9). 
-Frequencies 
Other Secondary/Exploratory Endpoints 
Percentage of patients requiring advanced 
oxygen support on Days 4 (±1), 8 (±1), 15 (±1), 
30 (±2,) and 60 (±3). 
-Frequencies 
-Unstratified odds ratio 
 


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Endpoints 
Analyses 
Time to intensification of respiratory support 
(WHO >5) (See Appendix 1 – 11-Point World 
Health 
Organization 
(WHO) 
Clinical 
Progression Scale). 
-Kaplan-Meier (unstratified)  
-Cox regression (unstratified) 
Total duration of advanced oxygen support. 
-Summary statistics 
-U-Mann Whitney 
Percentage of patients requiring high-flow 
oxygen on Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), 
and 60 (±3). 
-Frequencies 
-Unstratified odds ratio 
 
Total duration of high-flow oxygen therapy 
per patient. 
-Summary statistics 
-U-Mann Whitney 
Percentage of patients requiring non-invasive 
mechanical ventilation on Days 4 (±1), 8 (±1), 
15 (±1), 30 (±2), and 60 (±3). 
-Frequencies 
-Unstratified odds ratio 
 
Total duration of non-invasive mechanical 
ventilation per patient. 
-Summary statistics 
-U-Mann Whitney 
Percentage of patients requiring invasive 
mechanical ventilation or ECMO on Days 4 
(±1), 8 (±1), 15 (±1), 30 (±2), and 60 (±3). 
-Frequencies 
-Unstratified odds ratio 
 
Total duration of invasive mechanical 
ventilation or ECMO per patient. 
-Summary statistics 
-U-Mann Whitney 
Percentage of patients requiring admission to 
ICU on Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 
60 (±3). 
 
-Frequencies 
-Unstratified odds ratio 
 
Total duration of intensive care unit (ICU) 
stay. 
-Summary statistics 
-U-Mann Whitney 
Time to onset of additional (i.e., not present 
at baseline) immune-modulating drugs. 
-Kaplan-Meier (unstratified)  
-Cox regression (unstratified) 


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Endpoints 
Analyses 
Percentage of patients receiving immune-
modulating drugs on Days 1, 4 (±1), 8 (±1), 15 
(±1), 30 (±2), and 60 (±3). 
-Frequencies 
-Unstratified odds ratio 
 
Time to onset of additional (i.e., not present 
at baseline) antiviral drugs. 
-Kaplan-Meier (unstratified)  
-Cox regression (unstratified) 
Percentage of patients receiving subsequent 
antiviral drugs (i.e., not present at baseline) 
on Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 
(±3). 
-Frequencies 
-Unstratified odds ratio 
 
Percentage of patients with a new infection 
by Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 
(±3). 
-Frequencies 
-Unstratified odds ratio 
 
Cumulative mortality (all-cause and related 
to COVID-19) by Days 4 (±1), 8 (±1), 15 (±1), 
30 (±2), and 60 (±3). 
-Frequencies 
-Unstratified odds ratio 
 
Percentage of patients in each study group 
who require subsequent admission within 30 
days of initial end of hospital care [up to day 
60(±3)], related to either COVID-19 or COVID-
19 therapy. 
-Frequencies 
-Unstratified odds ratio 
 
Change respect to Day 1 in the viral load of 
SARS-CoV-2, measured by RT-PCR, by Days 4 
(±1), 8 (±1), 15 (±1), 30 (±2), and 60 (±3), in 
each study group. 
-Summary statistics 
-Univariate test (Fisher exact test or U-Mann 
Whitney) 
-Mixed model 
Percentage of patients in each study group 
with undetectable viral load, by Days 4 (±1), 8 
(±1), 15 (±1), 30 (±2), and 60 (±3). 
-Frequencies 
-Unstratified odds ratio 
 
Time to either undetectable viral load of 
SARS-CoV-2 or > 2 logs reduction respect to 
Day 1. 
-Kaplan-Meier (unstratified)  
-Cox regression (unstratified) 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
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Endpoints 
Analyses 
Change respect to baseline in inflammatory 
biomarkers (C- reactive protein [CRP], 
procalcitonin, lactate dehydrogenase [LDH], 
ferritin, neutrophil-to-lymphocyte ratio, and 
multiplex cytokines assay, by Day 4 (±1), 8 
(±1), 15 (±1) and 30 (±2) and 60 (±3). 
-Univariate test (Fisher exact test or U-Mann 
Whitney) 
-Mixed model 
Change respect to baseline in individual 
serological assessments against SARS-CoV-2, 
by Days 30 (±2), and 60 (±3). 
-Univariate test (Fisher exact test or U-Mann 
Whitney) 
-Mixed model 
Change respect to baseline in individual T-cell 
response against SARS-CoV-2 by Days 30 (±2), 
and 60 (±3). 
-Univariate test (Fisher exact test or U-Mann 
Whitney) 
-Mixed model 
Change respect to baseline to Days 15 (±1), 
and 30 (±2) in chest X-ray findings (Brixia 
score, centrally assessed) (Appendix 9 – Brixia 
Score). 
-Univariate test (Fisher exact test or U-Mann 
Whitney) 
-Mixed model 
Percentage of patients requiring modification 
of the therapy (drugs, dose or schedule) for 
the underlying disease. 
-Frequencies 
-Unstratified odds ratio 
 
Time in which pre-scheduled therapies for 
the control of the underlying disease were 
not able to be administered. 
-Kaplan-Meier (unstratified)  
-Cox regression (unstratified) 
Risk for mortality / clinical deterioration 
according to individual clinical characteristics 
and laboratory observations present at 
baseline, scores of validated scales (e.g., 
ISARIC-4C), as well as features related to 
COVID-19 [including SARS-CoV-2 variant (if 
available), vaccination status, duration of the 
infection, 
and 
inflammation-related 
parameters [neutrophil-to-lymphocyte ratio 
(NLR), protein C reactive, interleukine-6 (IL-6) 
and lymphocytes], the underlying disease, 
and other co-morbidities. 
-Frequencies 
-Unstratified odds ratio 
 
Limited-sampling 
pharmacokinetics 
assessment (See Section 7.6). 
 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
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Endpoints 
Analyses 
PK-PD analysis. 
 
 
9. ADMINISTRATIVE SECTION 
9.1 
ETHICS  
This clinical trial will be conducted in accordance with the ethical principles that have their origin 
in the World Medical Association (WMA) Declaration of Helsinki (96) and will be consistent with 
GCP guidelines and pertinent regulatory requirements. 
The study personnel involved in conducting this trial will be qualified by education, training and 
experience to perform their respective task(s). 
The study will be conducted in compliance with the protocol. The protocol, any amendments, 
and the patient’s ICF will receive IEC/IRB approval/favorable opinion prior to initiation. The 
decision of the IECs/IRBs concerning the conduct of the study will be made in writing to the 
Investigator and a copy of this decision will be provided to the Sponsor before commencement 
of the study. 
The Investigator and/or the Sponsor is/are responsible for keeping the IEC/IRB informed of any 
significant new information about the study drug. 
All protocol amendments will be agreed upon by the Sponsor and the Investigator. 
Administrative changes of the protocol are minor corrections and/or clarifications that have no 
impact on the way the study is to be conducted. 
9.2 
MONITORING, AUDITING AND INSPECTING 
The study will be monitored by regular site visits and telephone calls to the Investigator by the 
clinical trial monitor designated by the CRO and supervised by the Sponsor. Source document 
verification may be performed remotely, if required. 
During site visits, the trial monitor should revise original patient records, drug records and 
document retention (study file). Additionally, the trial monitor should observe study procedures 
and will discuss any problems with the Investigator. 
Adequate time for these visits should be allocated by the Investigator. The Investigator should 
also ensure that the monitor is given direct access [as per International Conference on 
Harmonization (ICH) Topic E6 Guideline for Good Clinical Practice, GCP] to source documents 
(i.e., hospital or private charts, original laboratory records, appointment books, etc.) of the 
patient which support data entered in the case report forms, as defined in the ICH Topic E6 
Guideline for Good Clinical Practice. 
Systems and procedures will be implemented to ensure the quality of every aspect of the trial. 
At any time during the course or at the end of the study, the Clinical Quality Assurance 
Department of Pharma Mar S.A. or external auditors contracted by the Sponsor may conduct an 
onsite or remote audit visit to the centres (ICH-GCP). 


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Protocol Number: AV-APL-B-002-22 
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Participation in this trial implies acceptance of potential onsite or remote inspection by national 
or foreign Competent Authorities. 
9.3 
PATIENT INFORMED CONSENT 
The rights, safety and well-being of the trial patients are the most important considerations and 
should prevail over interests of science and society. 
The ICFs will include all elements required by ICH-GCP and applicable regulatory requirements. 
Prior to inclusion into the trial, the Investigator or a person designated by the Investigator, must 
provide the patient with one copy of the ICFs for the clinical trial. This copy must provide written 
full information about the clinical trial, in a language that is non-technical and easily understood. 
The Investigator should allow the necessary time for the patient or his/her legally acceptable 
representative, when required, to inquire about the details of the clinical trial; then, both ICFs 
must be freely signed and personally dated by the patient and by the person who conducted the 
Informed Consent discussion before the beginning of the study. The patient should receive a 
copy of both signed ICFs and any other written information provided to study patients prior to 
participation in the trial. 
During a patient's participation in the trial, any updates to the ICFs and any updates to the 
written information will be provided to him/her. 
If there is a need to obtain new consent from the patients, the Investigator or a person 
designated by the Investigator should inform the patients of any new information relevant to 
the patients’ willingness to continue participation in the study, before obtaining the written 
consent. 
9.4 
CONFIDENTIALITY/ PATIENTS IDENTIFICATION 
The collection and processing of personal data from the patients enrolled in this clinical trial will 
be limited to those data that are necessary to investigate the efficacy, safety, quality and 
usefulness of the study drug used in this trial. 
It is the Investigator’s responsibility that sufficient information on the identity of the patients 
will be retained. 
The trial monitor, the Sponsor’s auditor, the IECs/IRBs and the Competent Authorities should 
have direct access to all requested trial-related records, and agree to keep the identity of study 
patients confidential. 
The data must be collected and processed with adequate precautions to ensure confidentiality 
and compliance with applicable data privacy protection laws and regulations. 
Explicit consent for the processing of personal data will be obtained from the participating 
patient before data collection, if applicable, and this consent should also address the transfer of 
the data to other entities and countries. 
Pharma Mar, S.A. (as Sponsor´s Headquarter) shall comply with General Data Protection 
Regulation (GDPR) (EU) 2016/679 effective from 25 May 2018 (repealing Directive 95/46/EC 
GDPR of 24 October 1995), and applicable regulations on the protection of individuals with 
regards to the processing of personal data and on the free movement of such data.  


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
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CONFIDENTIAL 
 
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9.5 
ELECTRONIC CASE REPORT FORMS 
Electronic CRFs (e-CRFs) will be used to record all data for each patient. It is the responsibility of 
the Investigator to ensure that the e-CRFs are properly and completely filled in, in English. E-
CRFs must be completed for all patients who have given their informed consent and have been 
enrolled into the study. 
A patient’s source documentation is the patient’s records (including but not limited to 
physician/hospital notes, nurses notes, IMP preparation records including reconstitution and 
dilution, IMP administration records, patient-reported outcomes, etc.) and any original 
document, and as such they should be maintained at the study site. 
The data collected in the e-CRF will be entered into the Sponsor’s databases which comply with 
GDPR (EU) 2016/679 effective from 25 May 2018 (repealing Directive 95/46/EC GDPR of 24 
October 1995) on the protection of individuals with regard to the processing of personal data 
and on the free movement of such data.  
9.6 
INSURANCE 
The Sponsor will provide insurance or indemnity in accordance with the applicable regulatory 
requirements. 
9.7 
RETENTION OF RECORDS 
The Investigator/Institution should maintain trial documents according to ICH Guideline for 
Good Clinical Practice and as required by applicable regulatory requirements. 
Essential documents should be retained as per the aforementioned ICH guideline or for a longer 
period of time, if required by the applicable regulations.  
9.8 
USE OF INFORMATION AND PUBLICATION 
Before the investigators of this study submit a paper or abstract for publication or otherwise 
publicly disclose information concerning the study drug or products, Pharma Mar S.A. must be 
provided with at least 60 days to revise and approve the proposed publication or disclosure to 
ensure that confidential and proprietary data are protected. 
If Pharma Mar S.A. determines that patentable patient matter is disclosed in the proposed 
publication or disclosure, the publication or disclosure will be withheld for a period of time 
considered convenient. If the study is part of a multicentre study, the first publication of the 
study shall be made in conjunction with the presentation of a joint, multicentre publication of 
the study results with the investigators and the institutions from all appropriate sites that are 
contributing data, analysis and comments. However, if such a multicentre publication is not 
submitted within 12 months after conclusion, abandonment or termination of the study at all 
sites, the present study may be published individually in accordance with the procedure 
established above. 
The order of the coauthors will reflect the relative contribution of each one to study 
development and analysis. In general, the first author will be the Investigator who recruits the 
highest number of patients with information finally available for data analysis. Relevant Pharma 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
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CONFIDENTIAL 
 
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Mar S.A. personnel who have fully participated in the study must be considered for co-
authorship of the publication. 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 –22 March 2023                                                                               Page 110
 
 
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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 –22 March 2023                                                                               Page 111
 
 
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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 –22 March 2023                                                                               Page 112
 
 
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Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
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biweekly plitidepsin as second-line therapy for advanced or metastatic transitional cell carcinoma 
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Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 –22 March 2023                                                                               Page 114
 
 
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study (ADMYRE) of plitidepsin in combination with dexamethasone vs. dexamethasone alone in 
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EM, Pradillo Fernández V, et al. Outcomes and Clinical Characteristics of the Compassionate 
Use of Plitidepsin for Immunocompromised Adult Patients with COVID-19. 2023. 
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treated COVID-19 patients. Hepatology international. 2020;14(5):2. 
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Favipiravir for COVID-19: An Open-Label Control Study. Engineering (Beijing, China). 
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in COVID-19: potential mechanisms and impact. Immunotherapy Advances. 2022;1(1). 
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immunological abnormality in patients with COVID-19: Possible mechanisms. Scandinavian 
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severity of COVID-19: a descriptive and predictive study. Signal transduction and targeted 
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severe coronavirus disease 2019 (COVID-19) infections: A systemic review and meta-analysis. 
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of T Cells in Patients With Coronavirus Disease 2019 (COVID-19). Frontiers in immunology. 
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Persistent replication of SARS-CoV-2 in a severely immunocompromised patient treated with 
several courses of remdesivir. International journal of infectious diseases : IJID : official 
publication of the International Society for Infectious Diseases. 2021;104:2. 


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 –22 March 2023                                                                               Page 115
 
 
82. 
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Severe Acute Respiratory Syndrome Coronavirus 2 Replication in an Immunocompromised 
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n+Graphical+Models+using+MCMC+https%3A%2F%2Fcran.r-


Pharma Mar S.A. 
Protocol Number: AV-APL-B-002-22 
EudraCT number: 2022-002489-34 
 
ICON Study: PAMAAPL1-AVAPL1 
CONFIDENTIAL 
 
Protocol. Version 3.0 –22 March 2023                                                                               Page 116
 
 
project.org+%E2%80%BA+web+%E2%80%BA+packages+%E2%80%BA+rjags&aqs=edge..6
9i57.542j0j4&sourceid=chrome&ie=UTF-8. 
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https://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki/. 

SAP_001.pdf: 
Protocol Number: AV-APL-B-002-22 
Official Title: A Multicentre, Open label, Randomised, Controlled, Basket, Pragmatic, Phase II, 
Clinical and Translational Study to Determine the Efficacy and Safety of Plitidepsin versus Control 
in Immunocompromised Adult Patients with Symptomatic COVID-19 requiring Hospital Care 
(NEREIDA) 
NCT Number: NCT05705167 
Document Date: 20 March 2024






Statistical Analysis Plan (SAP)
Version Date: 20Mar2024
Sponsor: PharmaMar
Protocol no: AV-APL-B-002-22
Page 3 of 28
Strictly Confidential.  © 2024, PRA Health Sciences.  All rights reserved.
3.0 Table of Contents
1.0 Approvals ................................................................................................................................................1
2.0 Change History .......................................................................................................................................2
3.0 Table of Contents....................................................................................................................................3
4.0 Purpose...................................................................................................................................................4
5.0 Scope......................................................................................................................................................4
6.0 Introduction .............................................................................................................................................4
7.0 Study Objectives .....................................................................................................................................4
7.1 Primary Objective.............................................................................................................................4
7.2 Key Secondary Objective.................................................................................................................4
7.3 Secondary Objectives......................................................................................................................4
8.0 Study Design...........................................................................................................................................6
8.1 Rationale..........................................................................................................................................6
8.2 Overall Design .................................................................................................................................6
8.3 Endpoints .........................................................................................................................................9
8.4 Randomisation...............................................................................................................................11
8.5 Sample Size Calculation................................................................................................................11
8.6 Population Sets..............................................................................................................................12
8.6.1 Replacement of Patients .....................................................................................................13
8.6.2 Populations..........................................................................................................................13
9.0 Conventions and Derivations................................................................................................................13
9.1 Imputation in Incomplete Dates .....................................................................................................14
10.0 Independent Data Monitoring Committee ...........................................................................................14
11.0 Statistical Methods..............................................................................................................................14
11.1 Subject disposition.......................................................................................................................15
11.2 Demographic and Baseline Characteristics.................................................................................15
11.3 Treatment Administration.............................................................................................................15
11.4 Protocol Deviations......................................................................................................................15
11.5 Concomitant medications and procedures ..................................................................................16
11.6 Efficacy Analyses.........................................................................................................................16
11.6.1 Primary Estimand ..............................................................................................................17
11.6.2 Key Secondary Estimand ..................................................................................................18
11.7 Safety Analyses ...........................................................................................................................18
11.7.1 Adverse Event of Special Interest .....................................................................................19
11.7.2 IDMC safety report.............................................................................................................20
11.8 Summary of Statistical Tests .......................................................................................................20
11.9 Vital signs, Physical Examinations, ECGs, Chest Imaging and Other Observations Related to 
Safety...................................................................................................................................................26
11.10 Pooled and Subgroup Analyses ................................................................................................26
11.11 Pharmacokinetic and Pharmacodynamics Analyses.................................................................26
12.0 References..........................................................................................................................................26
13.0 Statistical Software..............................................................................................................................27
14.0 Glossary of Abbreviations ...................................................................................................................27


Statistical Analysis Plan (SAP)
Version Date: 20Mar2024
Sponsor: PharmaMar
Protocol no: AV-APL-B-002-22
Page 4 of 28
Strictly Confidential.  © 2024, PRA Health Sciences.  All rights reserved.
4.0 Purpose
The Statistical Analysis Plan (SAP) describes the statistical methods to be used during the abbreviated 
reporting and analyses of data collected under PharmaMar Protocol AVL-APL-B-002-22.
5.0 Scope
The Statistical Analysis Plan outlines the following:
Study Objectives
Study Design
Study Estimands
Applicable Study Definitions
Statistical Methods
6.0 Introduction
This SAP should be read in conjunction with the study protocol and case report form (CRF). Any further 
changes to the protocol or CRF may necessitate updates to the SAP. A first version of the SAP is expected
prior to first patient in (FPI) and final approval of the SAP will occur prior to database lock. Due to low 
recruitment, early study closure was held on 23Feb24. Therefore, SAP is abridged to provide the essential 
results for an abbreviated Clinical Study Report (CSR) where information related to safety and descriptive 
efficacy results will be described. Study objectives, endpoints and populations will be adapted accordingly 
in order to present the most relevant data.
Consequently, no formal statistical significance tests will be presented, although the descriptive tables will 
allow a numerical comparison of the characteristics and outcomes of the patients in the experimental and 
control arm across the subgroups. 
7.0 Study Objectives
7.1 Primary Objective
To evaluate efficacy of plitidepsin in pre-specified groups of immunocompromised patients with 
symptomatic COVID-19 requiring hospital care vs control in terms of mortality.
7.2 Key Secondary Objective
To describe efficacy of plitidepsin and the control in terms of viral clearance, in each group.
7.3 Secondary Objectives
Efficacy secondary objectives
o
To describe efficacy of plitidepsin and the control in terms of sustained end of hospital care, 
in each group.
o
To describe efficacy of plitidepsin and the control in terms of symptomatic improvement, in
each group.
o
To describe efficacy of plitidepsin and the control in terms of clinical status (11 category 
WHO Clinical Progression Scale), in each group.


Statistical Analysis Plan (SAP)
Version Date: 20Mar2024
Sponsor: PharmaMar
Protocol no: AV-APL-B-002-22
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o
To describe efficacy of plitidepsin and control in terms of the need of any kind of 
supplementary oxygen, in each group.
Safety secondary objectives
o
To describe safety/tolerability of plitidepsin and the control in terms of adverse events, 
adverse reactions and mortality, in each group.
o
To describe safety/tolerability of plitidepsin and the control in terms of abnormal laboratory 
parameters, in each group.
o
To describe safety/tolerability of plitidepsin and the control in terms of variations of vital 
signs, in each group.
Other secondary objectives
o
To describe efficacy of plitidepsin and the control in the need of any type of advanced 
oxygen support (high-flow nasal oxygen, extracorporeal membrane oxygenation (ECMO), 
or non-invasive or invasive mechanical ventilation), in each group.
o
To describe efficacy of plitidepsin and the control in the need of intensification of respiratory 
or intensive care support, in each group.
o
To describe efficacy of plitidepsin and the control in the need of intensification of 
pharmacological therapies for COVID-19, in each group.
o
To describe efficacy of plitidepsin and control in terms of superinfection, in each group.
o
To describe efficacy of plitidepsin and the control in terms of all-cause- and related to 
COVID-19-mortality, in each group.
o
To describe efficacy of plitidepsin and the control in terms of subsequent hospital 
admissions, in each group.
o
To describe efficacy of plitidepsin and the control in terms of the time course of viral load, 
in each group.
o
To describe efficacy of plitidepsin and the control in terms of the evolution of inflammatory 
markers, in each group.
o
To describe efficacy of plitidepsin and the control in terms of the immune response against 
SARS-CoV-2, in each group.
o
To describe efficacy of plitidepsin and the control in terms of radiological evolution, in each 
group.
o
To describe efficacy of plitidepsin and the control in terms of restoration of the therapy for 
the underlying disease, in each group.
o
To describe safety/tolerability of all pooled plitidepsin arms and all control arms in all the 
aforementioned endpoints (all objectives).
o
To compare efficacy and safety/tolerability between plitidepsin arms (across different 
groups). <Not applicable as sample size does not allow any kind of comparison> 
o
To explore prognostic/predictive factors for clinical deterioration or mortality or drug 
response. <Not applicable as sample size does not allow any kind of comparison>


Statistical Analysis Plan (SAP)
Version Date: 20Mar2024
Sponsor: PharmaMar
Protocol no: AV-APL-B-002-22
Page 6 of 28
Strictly Confidential.  © 2024, PRA Health Sciences.  All rights reserved.
o
To increase pharmacology knowledge of plitidepsin.
8.0 Study Design
8.1 Rationale
Coronavirus disease 2019 (COVID-19), produced by the severe acute respiratory syndrome coronavirus 
type 2 (SARS-CoV-2), has caused a global pandemic. 
Immunocompromised patients are at increased risk for severe and fatal coronavirus disease 2019 
(COVID-19) outcomes. 
Patients with immune-system disorders have not been sufficiently represented in the clinical trials 
for COVID-19 antiviral drugs. 
Concerns have been raised about whether the accepted clinical practice standard to treat COVID-
19 with weak antiviral agents and immunosuppressive therapies may promote chronic infection 
and worse outcomes in immunosuppressed patients (at risk for protracted SARS-CoV-2 infection), 
including selection and transmission of new SARS-CoV-2 variants. 
Studies among immunocompromised hosts should ideally be focused on identifying direct-acting 
antivirals that markedly reduce SARS-CoV-2 replication, be they small molecules or antibodies.
Evidence of potent antiviral activity of plitidepsin observed in vitro and/or in vivo.
Plitidepsin anti-viral activity is “agnostic” to the different SARS-CoV-2 variants.
There is no signal supporting clinically-significant immunosuppressive potential of plitidepsin 
neither in the context of oncology clinical safety analysis after the first infusion of treatment nor in 
the clinical COVID-19 development (APLICOV-PC study).
The compassionate use of plitidepsin in immunosuppressed patients has shown:
o
Feasibility of plitidepsin in this target population of dismal prognosis.
o
Good tolerability of the 2.5 mg daily for three consecutive days.
o
Feasibility of a second salvage stint of plitidepsin in a small subset of patients with 
persistent viral load.
o
A median time to SARS-CoV-2 real time polymerase chain reaction (RT-PCR) 
negativisation of 17 days from plitidepsin Day 1.
o
A median time to oxygen independence of 13 days and, in line with this, significant 
radiological improvement of COVID-19 pneumonia, with exceptional requirement of high-
flow oxygen intervention or intensive care unit, frequently limited in this patient population.
A full rationale for the study may be found in the appropriate sections of the study Clinical Protocol.
8.2 Overall Design
This is a multi-centre, randomised, controlled, open-label, pragmatic, phase 2 basket study to assess the 
efficacy and safety of plitidepsin in different groups of immunocompromised patients with coronavirus 
disease 2019 (COVID-19) who require hospital care.
More than 50 sites are planned to participate in this study worldwide.
Approximately 150 patients are foreseen for the study, divided in 4 different groups of 
immunocompromised patients:
Group 1 –Patients receiving immune-suppression due to haematopoietic or organ transplantation.


Statistical Analysis Plan (SAP)
Version Date: 20Mar2024
Sponsor: PharmaMar
Protocol no: AV-APL-B-002-22
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Group 2 – Patients receiving B-cell depleting therapies.
Group 3 – Patients receiving other immune-suppressive therapies.
Group 4 – Other situations with immune deficiencies.
Whenever a patient can be allocated to more than 1 group, the priority will be group 1 > group 2 > group 
3 > group 4. In groups 1, 2, and 3, patients will be randomised to receive best standard care (BSC) ± other 
antiviral (if clinically indicated) on control arm and BSC + plitidepsin on experimental arm. Group 4 will not 
be controlled (it was not applicable in Spain), as shown in Figure 8.2.1.
Figure 8.2.1. Algorithm for Patient Assignment in NEREIDA Basket Trial.
Stratification:
Group 1: ISARIC mortali
intestinal/other)
Group 4: Not applicable


Statistical Analysis Plan (SAP)
Version Date: 20Mar2024
Sponsor: PharmaMar
Protocol no: AV-APL-B-002-22
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Study periods:
The study will include a Screening period, a Treatment period and a Follow-up period.
Refer to the Schedule of Assessments (Protocol Appendix 13) for data to be collected.
The Screening period starts once the patient has provided written informed consent and ends when the 
patient is randomized (allocated in group 4) in the study. Parameters assessed during this time will serve 
as the baseline values. The screening procedures should be preferably performed within 24 h prior to 
administration of study treatment, allowing a maximum of 48 h prior to administration of study treatment if 
required.
The Treatment period starts on Day 1 (date of randomisation) to 24 h after the last dose of the antiviral
therapy; if no antiviral was given in the control arm, then the treatment period will finish on Day 4, for data 
management purposes. Antiviral treatment must always be started on the randomisation date.
The Follow-up period starts after the end-of-treatment and lasts until the end-of-study.
The End of Study (EOS) is Day 60 (±3) or date of early study termination unless ongoing SAEs, if 
applicable. 
The End of Trial (EOT) will be when the Last Patient Last Visit (LPLV) has occurred.
All patients will receive the best available care as per applicable local, institutional, national, supranational 
COVID-19 treatment guidelines.
Patients assigned to the experimental arms will receive, in addition, plitidepsin as a 1-h IV infusion, during 
Days 1 to 3.
Patients randomised to the control arm may receive a regulatory-approved antiviral treatment in addition 
to the BSC.
For prevention of infusion-related (i.e., plitidepsin-related) reactions (IRRs), allergic reactions and emesis 
reactions, administration of premedications is mandatory in the experimental arms.
All consenting patients discontinuing early from the study before the EOS visit should complete the early 
termination/withdrawal procedures.
Duration of Study Periods:
Screening period: From the day of ICF signing prior to randomisation).
Day 1: Date of randomisation. Treatment must be started on the same date.
Treatment period: from Day 1 to 24 h after the last dose of the antiviral therapy; if no antiviral was 
given in the control arm, then the treatment period will finish on Day 4, for data management 
purposes.
Follow-up period: From the end-of-treatment to end-of-study.
EOS: Day 60 (±3) or date of early study termination unless ongoing SAEs, if applicable.
Overall study duration: 60 (±3) days since randomisation.
End of the trial will be when the Last Patient Last Visit (LPLV) has occurred. 
Independent Data Monitoring Committee (IDMC):
An IDMC was established to provide study oversight. The IDMC was composed of individuals external not 
only to the Sponsor but also to the trial managers (including Sponsor’s and Contract Research 
Organisation’s [CRO] medical monitors) and study investigators.
Given the decision to terminate the study due to low accrual, the IDMC activity has been discontinued and 
there will not be distinction between blinded and unblinded procedures.


Statistical Analysis Plan (SAP)
Version Date: 20Mar2024
Sponsor: PharmaMar
Protocol no: AV-APL-B-002-22
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Study “Stopping rules”
The Sponsor may suspend or terminate the study if:
New toxicological or pharmacological findings or safety issues that invalidate the earlier positive 
benefit-risk assessment occur;
Significant safety concerns, according to the Sponsor’s criteria, arise during the conduct of the 
study;
The development of plitidepsin is discontinued;
Any other reason (e.g., best interest of the patients, accrual rate makes the completion of the 
study feasible, etc.) leads to this suspension or termination.
The Sponsor has decided to terminate the study due to low accrual rate. 
Blinding/Unblinding 
For randomization purposes, no statistical personnel can access the RTSM Rave system.
For IDMC procedures, an independent team outside the scope of this SAP was created following the 
IDMC Charter.
For the clinical database, PharmaMar Biostats team remains blinded until the results of the analysis are 
released. Meanwhile, blinded outputs and datasets will be created by ICON Statistics & Programming
department for PharmaMar. The procedure planned to blind data for dry-run review consisted of shuffling
data and not including outputs with any CRF information where treatment arm is detailed (see below the 
specific CRF forms). However, given the early termination of the study, there is no risk of bias in the 
knowledge of randomization lists by the study personnel once the recruitment period has ended. 
Therefore, on 19/March/2024 it was decided to unmask data and continue with unblinded procedures. 
8.3 Endpoints
Efficacy primary endpoint: 
-
1-month† all-cause mortality rate. †(Day 30 since randomisation).
Key secondary endpoint:
-
Time to confirmed negativisation in SARS-CoV-2 antigen test or RT-PCR Ct > 30.
Secondary endpoints: 
Efficacy secondary endpoints
-
Time to sustained end of COVID-related hospital care from the time of randomisation. Sustained 
discharge is defined as no subsequent admission within 30 days of initial end of hospital care 
[up to day 60 (±3)], related to either COVID-19 or COVID-19 therapy.
-
Time to sustained improvement (defined in Protocol Section 7.1.3) and resolution of selected 
COVID signs/symptoms (See Protocol Appendix 10 - COVID-19 signs/symptoms checklist).
-
Distribution of patients according to their clinical status by the 11-category WHO Clinical 
Progression Scale, at Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 (±3) (See Protocol Appendix 
1 – 11-Point World Health Organization (WHO) Clinical Progression Scale).
-
Percentage of patients requiring oxygen therapy on Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 
(±3).
-
Time to sustained discontinuation (i.e., at least 7 days) of oxygen supplementation.


Statistical Analysis Plan (SAP)
Version Date: 20Mar2024
Sponsor: PharmaMar
Protocol no: AV-APL-B-002-22
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Safety secondary endpoints
Frequency of the following events (all-cause and drug-related):
-
Treatment-emergent adverse events (TEAEs);
-
-Common Terminology Criteria 
for AEs (CTCAE v.5.0);
-
Adverse events of special interest (AESIs);
-
Serious adverse events (SAEs);
-
Drug related Serious Adverse Events (i.e., SARs);
-
Adverse events leading to treatment discontinuation; and
-
Deaths (COVID-19-related/all).
-
Change respect to baseline* in individual study-defined laboratory parameters (See Protocol 
Section 5.7, 5.8 and 5.9 and Protocol Appendix 8 – Clinical Laboratory Analyses).
-
Change respect to baseline* in individual vital signs (See Protocol Section 5.7, 5.8 and 5.9).
*Baseline = latest test performed between screening and drug administration
Other Secondary/Exploratory Endpoints
-
Percentage of patients requiring advanced oxygen support on Days 4 (±1), 8 (±1), 15 (±1), 30 
(±2,) and 60 (±3).
-
Time to intensification of respiratory support (WHO >5) (See Protocol Appendix 1 – 11-Point 
World Health Organization (WHO) Clinical Progression Scale).
-
Total duration of advanced oxygen support.
-
Percentage of patients requiring high-flow oxygen on Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 
60 (±3).
-
Total duration of high-flow oxygen therapy per patient.
-
Percentage of patients requiring non-invasive mechanical ventilation on Days 4 (±1), 8 (±1), 15 
(±1), 30 (±2), and 60 (±3).
-
Total duration of non-invasive mechanical ventilation per patient.
-
Percentage of patients requiring invasive mechanical ventilation or ECMO on Days 4 (±1), 8 
(±1), 15 (±1), 30 (±2), and 60 (±3).
-
Total duration of invasive mechanical ventilation or ECMO per patient
-
Percentage of patients requiring admission to intensive care unit (ICU) on Days 4 (±1), 8 (±1), 
15 (±1), 30 (±2), and 60 (±3).
-
Total duration of ICU stay.
-
Time to onset of additional (i.e., not present at baseline) immune-modulating drugs.
-
Percentage of patients receiving immune-modulating drugs on Days 1, 4 (±1), 8 (±1), 15 (±1), 30 
(±2), and 60 (±3).
-
Time to onset of additional (i.e., not present at baseline) antiviral drugs.
-
Percentage of patients receiving subsequent antiviral drugs (i.e., not present at baseline) on 
Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 (±3).
-
Percentage of patients with a new infection by Days 4 (±1), 8 (±1), 15 (±1), 30 (±2), and 60 (±3).
-
Cumulative mortality (all-cause and related to COVID-19) by Days 4 (±1), 8 (±1), 15 (±1), 30 
(±2), and 60 (±3).


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-
Percentage of patients in each study group who require subsequent admission within 30 days of 
initial end of hospital care [up to Day 60(±3)], related to either COVID-19 or COVID-19 therapy.
-
Change respect to Day 1 in the viral load of SARS-CoV-2, measured by RT-PCR, by Days 4 
(±1), 8 (±1), 15 (±1), 30 (±2), 45 (±2), and 60 (±3), in each study group.
-
Percentage of patients in each study group with undetectable viral load, by Days 4 (±1), 8 (±1), 
15 (±1), 30 (±2), 45 (±2), and 60 (±3).
-
Time to either undetectable viral load of SARS-CoV-2 or > 2 logs reduction respect to Day 1
-
Change respect to baseline* in inflammatory and immunological (C- reactive protein [CRP], 
procalcitonin, lactate dehydrogenase [LDH], ferritin, neutrophil-to-lymphocyte ratio, absolute 
lymphocyte count (ALC), D-dimer, and multiplex cytokines assay, by Day 4 (±1), 8 (±1), 15 (±1) 
and 30 (±2) and 60 (±3).
-
Change respect to baseline* in individual serological assessments against SARS- CoV- 2, by 
Days 30 (±2), and 60 (±3).
-
Change respect to baseline* in individual T-cell response against SARS-CoV-2 by Days 30 (±2), 
and 60 (±3).
-
Change respect to baseline* to Days 15 (±2), and 30 (±2) in chest X-ray findings (Brixia score, 
centrally assessed) (Protocol Appendix 9 – Brixia Score).
-
Percentage of patients requiring modification of the therapy (drugs, dose or schedule) for the 
underlying disease.
-
Time in which pre-scheduled therapies for the control of the underlying disease were not able to 
be administered.
-
Limited-sampling pharmacokinetics assessment (See Protocol Section 7.6).
-
Pharmacokinetic(s)/Pharmacodynamic(s) (PK-PD) analysis
*Baseline = latest test performed between screening and drug administration
8.4 Randomisation
Patients will be sequentially allocated in their respective group, following the algorithm depicted in Figure 
8.2.1.
Central randomisation will be implemented by means of RTSM Rave, for full details on how the 
randomization lists are created and block selection see the randomization plan. In groups 1-3, patients 
will be assigned to the experimental or the control arms at a 1:1 ratio. Stratification will be performed in 
the randomised groups. The ISARIC-4C mortality score will be used as a stratification factor in all groups 
used 
for Group 1, whereas the underlying disease will be considered for Groups 2 and 3. For Group 2, the 
factor categories will be haematological neoplasm vs other disease; the respective categories for Group 3 
will be neoplasia vs other. Stratification is not applicable in the non-randomised Group 4.
Day 1 is defined as the day of randomisation. Treatment must start within the same natural day.
8.5 Sample Size Calculation 
A) Randomised groups (1-3)
Patients in three of the four groups will be randomly assigned to one of two arms. Each randomised group 
will have a sample size of 40 patients (20 in the experimental arm, 20 in the control arm).
Given the exploratory nature of this phase II study, this sample size has been chosen based on 
operational considerations to allow the feasibility of accrual in this relatively small population and also 


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provide preliminary estimates to establish an updated prior distribution of the mortality rates for further 
investigation using Bayesian methods in a potential Phase III study.
The operating characteristics of the calibrated Bayesian hierarchical model (CBHM) described by Chu 
and Yuan (1) have been investigated by means of the CBHM package from Yuan at MD Anderson 
Cancer Center (2), and with reference to the JAGS program running in R version 4.2.1 (3) on Microsoft 
Windows 10.
Within the CBHM simulations, 5000 Markov-chain Monte Carlo (MCMC) repetitions per scenario were 
simulated with a range of operating characteristics for the null, alternative and true mortality rates moving 
from 5% to 95% in steps of 5% (Table 1). A maximum sample size of 40 per randomised group was set 
through the CBHM simulations, which in turn determines whether there is a need for the total sample size 
to be allocated. Within all simulations, the full 120 patients were used, suggesting that this is the minimum 
sample size that enables decision-making. As an example, if the mortality rate in each group is 20% and 
we target a clinically meaningful absolute reduction of 12.5% in the experimental arm (H0=20%, 
H1=7.5%), and the assumed true mortality rate is 7.5%, the simulation demonstrates that a sample size 
of 40 per basket will allow for a posterior probability around 0.9 in each basket (i.e. 90% probability that 
the experimental arm is better than the control in each basket), ranging from 0.87 (when the data from the 
fastest basket is analysed) to 0.915 (at the end of the accrual of the last basket) (type I error rate= 0.05).
Table 1 Example of simulated posterior probabilities for sample size definition.
Mortality rate 
–control arm 
(null)
Mortality rate –
experimental arm 
(alternative)
True 
mortalit
y rate
Sample 
size per 
basket
Posterior 
probability 
1st basket
Posterior 
probability 
2nd basket
Posterior 
probability 
3rd basket
20%
7.5%
7.5%
30
0.82
0.84
0.865
20%
7.5%
7.5%
40
0.87
0.88
0.915
20%
7.5%
7.5%
50
0.895
0.905
0.93
20%
7.5%
7.5%
60
0.905
0.925
0.97
20%
7.5%
7.5%
70
0.945
0.98
0.985
20%
7.5%
7.5%
80
0.96
0.985
0.985
B) Non-controlled group 4
Clinical conditions that cannot be classified into one of the 3 randomised groups will be pooled into a 
fourth group of 30 patients. As this group is likely to be characterized by high heterogeneity, it will not 
have a control arm and will provide safety data on plitidepsin.. A Fleming’s phase II procedure with a 1-
stage design has been used to estimate the sample size needed to rule out that plitidepsin induces drug-
related SAEs in at least 35% of the patients (null hypothesis), and to confirm that the respective rate is 10 
% or lower (alternative hypothesis), with a type I error of 0.05 and a power of 90%.
If at least 25 patients out of a total sample of 30 do not experience a drug-related SAE, the null hypothesis 
will be rejected.
8.6 Population Sets
Immunocompromised adult patients with symptomatic COVID-19 requiring hospital care.
Patients must fulfill all the inclusion/exclusion criteria to be eligible to participate in the study.


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8.6.1 Replacement of Patients
No patients will be replaced. For each group 1-3, all efficacy analyses will be done on Full Analysis Set 
(FAS) population (See 8.6.2.1 below). 
Therefore, accrual in each group will end when the target number of evaluable patients in the FAS 
population has been reached.
8.6.2 Populations
The design of the trial, the Bayesian analysis and the sample size is based on the 30-day (+/- 2 d) survival 
information on the FAS population. The analysis of the primary and other time-to-event efficacy endpoints 
will be performed primarily in the FAS but also in the Per Protocol and ‘As treated’ populations. Supportive 
analyses will be performed on the Per Protocol (PP) population. Safety endpoints analyses will be based 
on the “As treated” population (also known as safety population). If there is any patient randomized by error 
in a different cohort that its correspondent cohort, for the analysis this patient will be moved 
programmatically to the valid one.
8.6.2.1 Full Analysis Set (FAS) Population
All randomised patients who have taken at least 1 dose of study treatment (plitidepsin or control) and 
have completed follow-up for survival until day 30 (±2). Patients who die before the end of follow-up 
period will also be included in the FAS population. FAS population will be analysed according to their 
randomised treatment arm.
8.6.2.2 As Treated Population
All patients who received any exposure to study treatment (plitidepsin or control). As Treated population 
will be analysed according to the treatment they actually received.
Day 1 therapy is defined as any exposure to study treatment (partial or complete).
<NOTE: Due to the premature study closure no PP specific outputs or sensitivity analyses will be 
generated in populations other than the FAS for baseline and efficacy analyses and safety population for 
safety analyses>.
9.0 Conventions and Derivations
Variables reported with different units will be homogenized to standardized variables following the 
International System of Units (e.g. laboratory tests, biometrical assessments...) unless otherwise specified.
By default, all results will be rounded to one decimal place, except when variables are integer, which will 
be reported without decimals (e.g., age in years). Percentages for 0 counts will not be presented and 100% 
values will be presented with no decimal places. For representing p-values four decimals will be selected 
as default but they could be rounded to fewer decimals if necessary. 
Missing values will not be included in the calculation of outputs. Unless otherwise noted, missing data will 
not be imputed or carried forward.
For patients who discontinue treatment due to adverse events (related or not to study treatment) every 
effort should made to remain under study follow-up, anyway, if information is lost before day 28 the 
conservative value of death will be also imputed. 


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Patients with no available data for any time-to event efficacy endpoint will be censored at time 0. EOS: Day 
60 (±3) or date of early study termination, if applicable. Patients not yet achieving the time to event endpoint 
will be censored at the last valid assessment prior to EOS. Efron’s method will be used to handle ties.
Assessment windows as specified in the clinical protocol will be respected, see protocol Appendix 13 
‘Schedule of Assessments and Procedures’ for definitions of trial days. Baseline is defined as latest test 
performed between screening and drug administration.
Change from baseline (%) is defined as: (test value-baseline value)/baseline value x 100.   
9.1 Imputation in Incomplete Dates
Dates of certain historical or current clinical activities are key component for statistical analysis. An 
incomplete date results from a missing day, month or year; in that case, the missing figure can be imputed 
allowing for the calculation of variables, such duration and time to certain event. However, when all of them, 
day, month and year, are missing no imputation will be done. 
Before randomisation/treatment start date
All variables needed to summarize for example prior information where partial information is available will 
be subject of imputation by means of SAS programming. If the day of a date is unknown then the imputed 
day will be 1, if the month is also unknown then the imputed date will 1/July. This assumption will be valid 
if the imputed date is earlier than the randomisation date; otherwise, the imputed date will be the first day 
of the month of the randomisation date (i.e. 01/Randomisation month date/year).
Between treatment start and end of treatment
All date variables during treatment where information is needed and is not fully available, for example 
adverse events or concomitant medications, will be subject of imputation by means of SAS programming. 
If the day of a date is unknown then the imputed day will be 1, if the month and/or year is also unknown 
then the imputed date will be 1/January (this assumption will be valid if the imputed date is earlier than the 
treatment start date; otherwise, the imputed date will be the treatment start date). For treatment emergent 
AEs/CMs a conservative approach will be taken and if imputed date may be considered treatment emergent 
or medication taken on treatment then always will be counted.
After end of treatment
A conservative approach for the variables collecting information after end of treatment where partial 
information is available will be imputed by means of SAS programming. The following rules will be 
implemented: if the day of a date is unknown then the imputed day will be 1; if the month is also unknown, 
then the imputed date will be 1/July. This assumption will be valid if the imputed date occurs later than the 
last drug administration date; otherwise, the imputed date will be the last drug administration date plus 1 
day. 
10.0 Independent Data Monitoring Committee
An IDMC was established to provide study oversight. The IDMC was established and operated in 
compliance with the FDA Guidance for Industry “Establishment and Operation of Clinical Trial Data 
Monitoring Committees”.
A separate IDMC charter has been written in order to specify the whole IDMC study conduct.
11.0 Statistical Methods
Non-continuous variables will be described in frequency tables using counts and percentages. Continuous 
variables will be described by median, mean, standard deviation (STD), interquartile range (Q1-Q3), 


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minimum and maximum. Study endpoints will be summarized by means of tables, listings for all CRF 
variables and figures for temporal series as viral load, lymphocytes/neutrophils evolution, LDH, CT>30 or 
oxygen necessities as well as Kaplan-Meier figures, as inverse KM (see section 11.8) where time to event 
data Y-axis started from 0% instead the usual 100%, e.g. time to confirmed negativisation in SARS-CoV-2 
antigen test or RT-PCR Ct > 30, will be shown.
11.1 Subject disposition
Main characteristics concerning inclusion in the study, randomisation, enrolment by country/site, end of 
treatment and withdrawal of the study will be displayed in this section.
11.2 Demographic and Baseline Characteristics
Baseline data such as for example demographics, medical history, clinical evaluation will be described in 
the FAS population.
For pre-treatment characteristics with multiple measurements per patient before the start of treatment (e.g.
laboratory assessments, vital signs), the baseline measurement will be considered the last value prior to or 
on the first day of treatment. Continuous variables that will be also presented as categorized summaries.
Age at informed consent will be summarized as continuous variable and categorized as <65y, >=65-74y
and >=75y. Gender, race, stratification factors from eCRF randomization forms and from their 
corresponding eCRF forms, physical examination form including height, weight, BMI and abnormal results, 
respiratory assessments form including oxygen therapy status, SpO2, FiO2, SpO2/FiO2, PaO2 and 
PaO2/FiO2,vital signs form including systolic blood pressure, diastolic blood pressure, pulse and respiratory 
rate, ECG Test Results - Local Reading form including heart rate, PR interval, QRS duration, QT interval, 
QTcF interval (Fridericia's formula) and QTcF (ECG measurements will be taken in triplicates but average 
values will be shown by visit), SARS-CoV-2 Test, COVID 19 Vaccination, duration of the infection prior to 
study entry defined as time from currently COVID-19 episode diagnosis date to randomization as 
continuous variable and categorized as <5 days and >=5 days and any additional variable considered as 
clinically relevant.
11.3 Treatment Administration
Treatment compliance defined as the extent to which patients take medications as requested in the protocol 
will be measured as duration in days of antiviral exposure ((Last exposure date-First exposure date)+1), 
total number of infusions/doses administered, the number of infusions/doses where total amount was 
administered, the number of infusions/doses where total amount was not administered and the number of 
infusions interrupted will be listed and summarized using descriptive statistics where applicable.
11.4 Protocol Deviations
A protocol deviation is defined as any departure from what is described in the protocol of a clinical trial. The 
study specific Protocol Deviation Guidance Document defines all important protocol deviations. 
The study team will conduct on-going reviews of the deviation data and document must be finalized prior 
to database lock.
Summaries of protocol deviations will be produced. 
Deviations with no effects on the risk/benefit ratio of the clinical trial (such as minimal delays in assessments 
or visits) will be distinguished from those that might have an effect on this risk/benefit ratio.
Below examples of deviation’s classification are listed. 




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The resultant posterior distribution will serve as a starting point for further trials within this area, whether 
as a key part of any information data packs provided to experts ahead of an elicitation meeting, or directly 
as the informed prior or hypotheses for further trials (5) (6).
A calibrated Bayesian hierarchical model (CBHM) described by Chu and Yuan (1) was planned to 
analyse the data, allowing data to be analysed by group and results from each group be used to enrich 
the data of the other groups. The calibration of the model allows strong information sharing when the 
treatment effect is similar among baskets, and shrinks that sharing towards 0 as the treatment effect 
becomes more heterogeneous.
Given the early termination of the study and the low recruitment in cohorts 1 and 3, the enrichment of 
groups with information from the other treatment groups will not be possible. Therefore, no CBHM will be 
performed for the main analysis and the information of the different cohorts will be combined, if 
applicable, only for safety analysis. The primary endpoint: 1-month† all-cause mortality rate will be 
studied by means of descriptive mortality rate analysis. 
Primary Endpoint
The difference between treatment and control arms in all-cause mortality at 30 days was foreseen to be 
examined using a Bayesian comparison of proportions within the calibrated Bayesian hierarchical model 
(CBHM) framework described by Chu and Yuan (1). 
As each basket completes, they would have been evaluated for efficacy, with information gained being 
used to update the analyses of successive baskets.
Due to the premature study closure no Bayesian specific outputs or sensitivity analyses will be generated
Secondary Endpoints/Other Endpoints
Frequency tables will be prepared for categorical variables, and continuous variables will be described by 
means of summary tables, which will include the median, mean, standard deviation, interquartile range, 
minimum, and maximum of each variable.
Frequentist methods will be calculated. Counts and percentages, with their corresponding exact 
confidence intervals, will be calculated for the binomial endpoints.
Time-to-event variables (e.g. key secondary endpoint) and their set time estimates will be defined as time 
from randomization to their corresponding event + 1 (see 8.3 section) and analysed according to the 
Kaplan-Meier method.
Key secondary endpoint, time to confirmed negativisation in SARS-CoV-2 antigen test or RT-PCR Ct > 
30, will be analysed according to the Kaplan-Meier method.
<NOTE: Due to the premature study closure no complex specific outputs or sensitivity analyses will be 
generated>.
11.6.1 Primary Estimand
The primary estimand corresponding to the primary endpoint is defined as:
Treatment: Participants will be randomised to receive plitidepsin versus control in each group.
Population: Participants as defined through the inclusion and exclusion criteria, who have been 
randomised to the trial and who have taken at least 1 dose of study treatment (plitidepsin or control).
Variable:
–1-month all-cause mortality rate (Day 30 since randomisation).


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Intercurrent events:
-
The participant’s data are collected and included in the analysis without regard for treatment 
discontinuation (treatment-policy strategy).
-
In the event of the participant initiating another non-protocol therapy, 1-month all-cause mortality 
rate will be evaluated regardless of initiation of new non-protocol therapy (treatment-policy 
strategy).
Population level summary: the posterior probability associated for the odds ratio of 1-month all-cause 
mortality rate from a Bayesian logistic regression for each group comparison including point estimate and 
credible interval will be shown.
The cut-off for considering further clinical development of plitidepsin in each randomized group will be a 
posterior probability of superiority for the experimental arm equal to or greater than 65%.
<NOTE: Due to the premature study closure no Bayesian specific outputs will be generated and 
descriptive data will be shown>.
11.6.2 Key Secondary Estimand
The key secondary estimand corresponding to the key secondary endpoint is defined as:
Treatment: Participants will be randomised to receive plitidepsin versus control in each group.
Population: Participants as defined through the inclusion and exclusion criteria, who have been 
randomised to the trial and who have taken at least 1 dose of study treatment (plitidepsin or control).
Variable:
–Time to confirmed negativisation in SARS-CoV-2 antigen test or RT-PCR Ct > 30 calculated as 
date/time of survival event – first dose date/time of Investigational Product administration.
Intercurrent events:
-
The participant’s data are collected and included in the analysis without regard for treatment 
discontinuation (treatment-policy strategy).
-
In the event of the participant initiating another non-protocol therapy, time to confirmed 
negativisation will be evaluated regardless of initiation of new non-protocol therapy (treatment-
policy strategy).
Population level summary: the hazard ratio from a Cox regression for each group comparison including 
point estimate and confidence interval will be shown.
<NOTE: Due to the premature study closure no complex specific outputs will be generated and 
descriptive data will be shown>.
11.7 Safety Analyses
Safety analyses will be based on the As Treated Population. All safety parameters will be summarised, 
plotted and also listed by patient/events.
A treatment-emergent adverse event is defined as any event not present prior to the initiation of the 
treatments or any event already present that worsens in intensity following exposure to the treatments.


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Treatment-emergent Adverse Events: The verbatim terms used in the electronic case report form (eCRF) 
by investigators to identify TEAEs will be coded using the Medical Dictionary for Regulatory Activities 
(MedDRA) and will be graded according to NCI CTCAE version 5.0. Treatment-emergent AEs will be 
summarised by MedDRA system organ class and preferred term by number of patients and by number of 
events. For each TEAE, the percentage of subjects who experience at least 1 occurrence of the TEAE will 
be summarised overall and by treatment group. The SAEs, AEs of special interest, and deaths will be listed
including the study period (see Section 8.2) that they occur. All TEAEs resulting in discontinuation of study 
treatment will be listed and summarised by system organ class/preferred term.
AEs, SAEs, deaths, laboratory evaluations, and study drug discontinuations due to AEs will be tabulated in 
a descriptive way. Counts and percentages will be used for categorical variables, and summary tables will 
be used for continuous variables.
Any treatment-related adverse events (including Unknown relationship but potentially related), treatment-
emergent adverse events of special interest, and SAES (including those with seriousness missing) that will 
measured daily until resolution or stabilisation to at least Grade 1, or to an acceptable level according to 
the investigator and the sponsor of his/her designated representative.
Clinical Laboratory Tests, see protocol sections 5.5.1/5.7/5.8/5.9 and appendix 8 to differentiate local or 
central. Laboratory data will be summarised by type of laboratory test. The worst toxicity grade will be 
tabulated. Parameters with predefined NCI CTCAE version 5.0 toxicity grades will be summarised. Change 
from baseline to the worst TEAE grade experienced by the subject during the study will be provided as shift 
tables.
Haematology: haematocrit, red blood cells (RBC), differential WBC counts (including neutrophil 
and lymphocyte counts), platelet count and haemoglobin.
Biochemistry: Liver function test [ALT, AP, AST, albumin, total bilirubin (direct bilirubin if total is 
abnormally elevated)], lactate dehydrogenase (LDH), glucose [fasting], sodium, potassium, 
calcium, BUN/urea, gamma glutamyl transferase (GGT), creatinine, calculated creatine clearance 
[Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)], CPK, amylase, lipase, pro-
calcitonin, ferritin, and CRP.
Coagulation (D-Dimer).
Urine analysis (semi-quantitative elemental tests for specific gravity, blood, pH, proteins, bilirubin, 
glucose, ketones, nitrites, and qualitative analysis of the sediment).
11.7.1 Adverse Event of Special Interest
The following AEs will be considered and monitored as AEs of special interest (AESIs): musculoskeletal 
disorders, CPK increases, and rhabdomyolysis; hypersensitivity reactions, cardiac events and 
transaminase elevations, thromboembolic events, and hepatobiliary disorders. 
If at any time AST/ALT levels are greater than 3 x ULN (> 5 x ULN if pre-existent liver involvement at 
baseline by the underlying disease, with baseline ALT  levels between 3-5 x ULN):
Report as an AESI.
The investigator should ensure that serum liver enzyme tests, serum bilirubin, and coagulation 
tests (INR or equivalent) are repeated two or three times weekly (local labs). Frequency of re-


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testing can decrease to once a week or less if abnormalities stabilise or the trial drug has been 
discontinued and the subject is asymptomatic. 
The list of concomitant medications should be re-checked, and include nonprescription drugs, 
herbal and supplementary preparations, alcohol and recreational substances. 
Other reasons for liver injury should be ruled out, including co-infection with other virus, 
autoimmune or alcoholic hepatitis, non-alcoholic liver steato-hepatitis, biliary tract disease, 
progression of an underlying disease (e.g. liver metastasis), hypoxic/ischemic hepathopathy, or 
exposure to environmental agents. 
The investigator should consider additional tests to evaluate liver function or structure, as well as 
a consultation to specialists in liver diseases.
Treatment with plitidepsin must be discontinued in case of:
ALT or AST > 8 x ULN;
ALT or AST > 3 x ULN and either total bilirubin >2 x ULN or INR >1.5;
ALT or AST > 3xULN with the appearance of fatigue, nausea, vomiting, right upper quadrant pain 
or tenderness, fever, rash, and/or eosinophilia (>5%).
11.7.2 IDMC safety report
IDMC requested specific outputs based on the publication (submitted): DMC reports in the 21st century. 
Towards better tools for decision-making, Vandemeulebroecke M., Baillie, M. Mirshani, A. and Lesaffre E.
Due to the premature study closure and suspension of the IDMC activity, no specific outputs or sensitivity 
analyses will be generated
11.8 Summary of Statistical Tests
All the main characteristics for estimands to fulfill study objectives will be defined as footnotes in each table 
following a similar structure than primary estimand definition. Below it can be found the endpoints to 
measure the variable definition and the analysis method to be used. Treatment attribute is maintained as 
‘participants will be randomised to receive plitidepsin versus control in each group’ whereas applicable and 
sensitivity analyses according populations defined in 8.5 will be calculated. For intercurrent events, while 
applicable, a treatment-policy strategy as defined for the primary estimand will be followed.
Table 3 Summary of statistical tests 
Endpoints
Analyses
Population
Efficacy primary endpoint
1-month all-cause mortality rate. 
-Frequencies
-FAS population
Key secondary endpoint
Time to confirmed negativisation
in SARS-CoV-2 antigen test or 
RT-PCR Ct > 30.
-Kaplan-Meier (unstratified) 
-FAS population


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Endpoints
Analyses
Population
Efficacy secondary endpoints
Time to sustained end of 
COVID-related hospital care
from the time of randomisation. 
Sustained discharge is defined 
as no subsequent admission 
within 30 days of initial end of 
hospital care [up to day 60 (±3)], 
related to either COVID-19 or 
COVID-19 therapy
-Kaplan-Meier (unstratified) 
-FAS population
Time to sustained improvement 
(defined in Protocol Section 
7.1.3) and resolution of selected 
COVID signs/symptoms (See 
Protocol Appendix 10 - COVID-
19 signs/symptoms checklist).
-Kaplan-Meier (unstratified) 
-FAS population
Distribution of patients according 
to their clinical status by the 11-
category WHO Clinical 
Progression Scale, at Days 4 
(±1), 8 (±1), 15 (±1), 30 (±2), 
and 60 (±3) (See Protocol
Appendix 1 – 11-Point World 
Health Organization (WHO) 
Clinical Progression Scale).
-Frequencies
-FAS population
Percentage of patients requiring 
oxygen therapy on Days 4 (±1), 
8 (±1), 15 (±1), 30 (±2), and 60 
(±3).
-Frequencies
-FAS population
Time to sustained 
discontinuation (i.e., at least 7 
days) of oxygen 
supplementation.
-Kaplan-Meier (unstratified) 
-FAS population
Safety secondary endpoints
Treatment-emergent adverse 
events (TEAEs).
-Frequencies (severity grade by 
NCI-CTCAE v5)
-As treated population
the National Cancer Institute 
-Frequencies
-As treated population


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Endpoints
Analyses
Population
[NCI]-Common Terminology 
Criteria for AEs (CTCAE v.5.0).
Adverse events of special 
interest (AESIs).
-Frequencies (severity grade by 
NCI-CTCAE v5)
-As treated population
Serious adverse events (SAEs).
-Frequencies (severity grade by 
NCI-CTCAE v5)
-As treated population
Drug related Serious Adverse 
Events (i.e., SARs).
-Frequencies (severity grade by 
NCI-CTCAE v5)
-As treated population
Adverse events leading to 
treatment discontinuation.
-Frequencies (severity grade by 
NCI-CTCAE v5)
-As treated population
Deaths (COVID-19-related/all).
-Frequencies
-As treated population
Change respect to baseline in 
individual study-defined 
laboratory parameters (See 
Protocol Section 5.7, 5.8 and 5.9
and Appendix 8 - Clinical 
Laboratory Analyses).
-Frequencies (severity grade by 
NCI-CTCAE v5)
-As treated population
Change respect to baseline in 
individual vital signs (See 
Protocol Section 5.7, 5.8 and 
5.9).
-Frequencies
-As treated population
Other Secondary/Exploratory Endpoints
Percentage of patients requiring 
advanced oxygen support on 
Days 4 (±1), 8 (±1), 15 (±1), 30 
(±2,) and 60 (±3).
-Frequencies
-FAS population
Time to intensification of 
respiratory support (WHO >5) 
(See Protocol Appendix 1 – 11-
Point World Health Organization 
(WHO) Clinical Progression 
Scale).
-Kaplan-Meier (unstratified) 
-FAS population
Total duration of advanced 
oxygen support.
-Summary statistics
-FAS population


Statistical Analysis Plan (SAP)
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Endpoints
Analyses
Population
Percentage of patients requiring 
high-flow oxygen on Days 4 
(±1), 8 (±1), 15 (±1), 30 (±2), 
and 60 (±3).
-Frequencies
-FAS population
Total duration of high-flow 
oxygen therapy per patient.
-Summary statistics
-FAS population
Percentage of patients requiring 
non-invasive mechanical 
ventilation on Days 4 (±1), 8 
(±1), 15 (±1), 30 (±2), and 60 
(±3).
-Frequencies
-FAS population
Total duration of non-invasive 
mechanical ventilation per 
patient.
-Summary statistics
-FAS population
Percentage of patients requiring 
invasive mechanical ventilation 
or ECMO on Days 4 (±1), 8 (±1), 
15 (±1), 30 (±2), and 60 (±3).
-Frequencies
-FAS population
Total duration of invasive 
mechanical ventilation or ECMO 
per patient.
-Summary statistics
-FAS population
Percentage of patients requiring 
admission to ICU on Days 4 
(±1), 8 (±1), 15 (±1), 30 (±2), 
and 60 (±3).
-Frequencies
-FAS population
Total duration of intensive care 
unit (ICU) stay.
-Summary statistics
-FAS population
Time to onset of additional (i.e., 
not present at baseline) 
immune-modulating drugs.
-Kaplan-Meier (unstratified) 
-FAS population
Percentage of patients receiving 
immune-modulating drugs on 
Days 1, 4 (±1), 8 (±1), 15 (±1), 
30 (±2), and 60 (±3).
-Frequencies
-FAS population
Time to onset of additional (i.e., 
not present at baseline) antiviral 
drugs.
-Kaplan-Meier (unstratified) 
-FAS population


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Endpoints
Analyses
Population
Percentage of patients receiving 
subsequent antiviral drugs (i.e., 
not present at baseline) on Days 
4 (±1), 8 (±1), 15 (±1), 30 (±2), 
and 60 (±3).
-Frequencies
-FAS population
Percentage of patients with a 
new infection by Days 4 (±1), 8 
(±1), 15 (±1), 30 (±2), and 60 
(±3).
-Frequencies
-FAS population
Cumulative mortality (all-cause 
and related to COVID-19) by 
Days 4 (±1), 8 (±1), 15 (±1), 30 
(±2), and 60 (±3).
-Frequencies
-FAS population
Percentage of patients in each 
study group who require 
subsequent admission within 30 
days of initial end of hospital 
care [up to day 60(±3)], related 
to either COVID-19 or COVID-
19 therapy.
-Frequencies
-FAS population
Change respect to Day 1 in the 
viral load of SARS-CoV-2, 
measured by RT-PCR, by Days 
4 (±1), 8 (±1), 15 (±1), 30 (±2), 
and 60 (±3), in each study 
group.
-Summary statistics
-FAS population
Percentage of patients in each 
study group with undetectable 
viral load, by Days 4 (±1), 8 (±1), 
15 (±1), 30 (±2), and 60 (±3).
-Frequencies
-FAS population
Time to either undetectable viral 
load of SARS-CoV-2 or > 2 logs 
reduction respect to Day 1.
-Kaplan-Meier (unstratified) 
-FAS population
Change respect to baseline in 
inflammatory biomarkers (C-
reactive protein [CRP], 
procalcitonin, lactate 
dehydrogenase [LDH], ferritin, 
neutrophil-to-lymphocyte ratio, 
and multiplex cytokines assay, 
-Summary statistics
-FAS population


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Protocol no: AV-APL-B-002-22
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Endpoints
Analyses
Population
by Day 4 (±1), 8 (±1), 15 (±1) 
and 30 (±2) and 60 (±3).
Change respect to baseline in 
individual serological 
assessments against SARS-
CoV-2, by Days 30 (±2), and 60 
(±3).
-Summary statistics
-FAS population
Change respect to baseline in 
individual T-cell response 
against SARS-CoV-2 by Days 
30 (±2), and 60 (±3).
-Summary statistics
-FAS population
Change respect to baseline to 
Days 15 (±1), and 30 (±2) in 
chest X-ray findings (Brixia 
score, centrally assessed) 
(Protocol Appendix 9 – Brixia 
Score).
-Summary statistics
-FAS population
Percentage of patients requiring 
modification of the therapy 
(drugs, dose or schedule) for the 
underlying disease.
-Frequencies
-FAS population
Time in which pre-scheduled 
therapies for the control of the 
underlying disease were not 
able to be administered.
-Kaplan-Meier (unstratified) 
-FAS population
Risk for mortality / clinical 
deterioration according to 
individual clinical characteristics 
and laboratory observations 
present at baseline, scores of 
validated scales (e.g., ISARIC-
4C), as well as features related 
to COVID-19 [including SARS-
CoV-2 variant (if available), 
vaccination status, duration of 
the infection, and inflammation-
related parameters [neutrophil-
to-lymphocyte ratio (NLR), 
protein C reactive, interleukine-6 
(IL-6) and lymphocytes], the 
underlying disease, and other 
co-morbidities.
-Frequencies
-FAS population


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Protocol no: AV-APL-B-002-22
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Endpoints
Analyses
Population
<NOTE: Due to the premature study closure no Bayesian or specific outputs or sensitivity analyses will 
be generated>.
11.9 Vital signs, Physical Examinations, ECGs, Chest Imaging and Other 
Observations Related to Safety
Vital signs, physical examination, ECGs, chest imaging and respiratory assessments data including oxygen 
therapy supplementation status and their changes from baseline will be summarized by visit using standard 
descriptive statistics for the as treated population.
11.10 Pooled and Subgroup Analyses
Exploratory pooled analyses will be performed for safety endpoints.
<NOTE: Due to the premature study closure no specific outputs will be generated for subgroup analysis>.
11.11 Pharmacokinetic and Pharmacodynamics Analyses
These analyses will be detailed and reported in separate documents.
12.0 References
1.
Y. C, Y. Y. A Bayesian basket trial design using a calibrated Bayesian hierarchical model. Clinical 
trials (London, England). 2018;15(2).
2.
Ying Yuan 2022 [Available from: https://odin.mdacc.tmc.edu/~yyuan/] 
3.
Bayesian Graphical Models using MCMC [Available from: https://cran.r-project.org]. Qinghua 
(Kathy) Chen. Incorporating Graphics into Summary Report Tables using ODS and GTL. 
PharmaSUG2011 – Paper SP02.
4.
G. P, Cro S, Mason A, Phillips R, Cornelius V. Design and analysis features used in small 
population and rare disease trials: A targeted review. Journal of clinical epidemiology. 2022;144:93-
101
5.
V. HL, Whitehead J, Eleftheriou D, Tudur-Smith C, Jones R, Jayne D, et al. Elicitation of expert 
prior opinion: application to the MYPAN trial in childhood polyarteritis nodosa. PloS one. 
2015;10(3):e0120981. 
6.
N. D, Best N, Montague TH. Better decision making in drug development through adoption of formal 
prior elicitation. Pharmaceutical statistics. 2018;17(4):301-16
7.
Berry SM, Broglio KR, Groshen S, et al. Bayesian hierarchical modeling of patient subpopulations: 
efficient designs of phase II oncology clinical trials. Clin Trials 2013; 10: 720–734.


Statistical Analysis Plan (SAP)
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Protocol no: AV-APL-B-002-22
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13.0 Statistical Software
JAGS program running in R version 4.2.1 (3) on Microsoft Windows 10 for simulations and sample size 
derivation for randomised groups (1-3) and https://www2.ccrb.cuhk.edu.hk/stat/phase2/Fleming.htm for 
Non-controlled group 4.
SAS® v.9.4 or higher will be used for statistical analysis outputs.
14.0 Glossary of Abbreviations
AE
Adverse Event
AESI
Adverse Event of Special Interest
ALC
Absolute Lymphocyte Count
ATC
Anatomical Therapeutic Chemical
BSC
Best Standard Care
CBHM
Calibrated Bayesian Hierarchical Model
CKD-EPI
Chronic Kidney Disease Epidemiology Collaboration
COVID-19
Coronavirus disease 2019
CRO
Contract Research Organisation
CRP
C- reactive Protein
CRF
Case Report Form
eCRF
Electronic Case Report Form
ECMO
Extracorporeal Membrane Oxygenation
EOS
End of Study
FAS
Full Analysis Set
FPI
First Patient In
GCP
Good Clinical Practice
GGT
Gamma Glutamyl Transferase
h
Hour
ICF
Informed Consent Form
ICU
  
Intensive Care Unit
IDMC
Independent Data Monitoring Committee
IL-6
Interleukine-6
IMP
Investigational Medicinal Product
IRR
Infusion-related (i.e., plitidepsin-related) Reactions
IV
Intravenous
JAGS
Just Another Gibbs Sampler
LDH
Lactate Dehydrogenase
LPLV
Last Patient Last Visit
MCMC
Markov-chain Monte Carlo
MedDRA
Medical Dictionary for Regulatory Activities
NCI-CTCAE
National Cancer Institute Common Terminology Criteria for Adverse Events
NLR
Neutrophil-to-Lymphocyte Ratio
PK/PD
Pharmacokinetic(s)/Pharmacodynamic(s)
PP
Per Protocol
Q1-Q3
Interquartile Range
RBC
Red Blood Cells
RT-PCR
Real Time Polymerase Chain Reaction
SAE
Serious Adverse Event


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SAR
Drug related Serious Adverse Event
SARS-CoV-2  
Severe Acute Respiratory Syndrome Coronavirus Type 2
STD
Standard Deviation
TEAE
Treatment Emergent Adverse Event
vs
versus
WHO
World Health Organization