Conditions: Colorectal Cancer
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 1
Healthy volunteers: no

Brief summary:
The current study will evaluate the microbiome-derived therapeutic vaccine EO2040 in combination with nivolumab in patients with circulating tumor DNA-defined Minimal Residual Disease (MRD) of colorectal cancer stage II, III, or IV after completion of standard curative therapy.

Detailed description:
The microbiome-derived therapeutic vaccine concept utilized in conjunction with anti-Programmed cell Death protein 1 (PD1) blockade is an innovative option for testing of a rational immunotherapy in colorectal cancer. The concept as such, including the combination with nivolumab, has already been tested in the clinical setting (i.e. in recurrent glioblastoma and adrenal tumors) and shown to be well tolerated.

Arms:
arm 1: Patients With Circulating Tumor DNA-defined Minimal Residual Disease of Colorectal Cancer Stage II, III, or IV After Completion of Curative Therapy

Interventions: intervention 1: EO2040
Intervention descriptions: intervention 1: EO2040, is a therapeutic peptide vaccine composed of two microbial-derived peptides mimicking cytotoxic T cell (CD8+ T cell) epitopes from the Tumor Associated Antigens (TAAs) combined with the helper peptide (CD4+ T cell epitope) Universal Cancer Peptide 2 (UCP2).

The peptide mix EO2040, i.e. drug product (DP), will be emulsified with the adjuvant Montanide.

EO2040 will be given in combination with nivolumab, which is an anti-PD1. Nivolumab is approved for use for the treatment of multiple cancer types, including subtypes of CRC (mismatch repair deficient or microsatellite instability-high metastatic disease after prior treatment). However, it is not currently approved for ctDNA defined MRD of CRC.

Protocol document:
SAP_002.pdf: 
 _____________________________________________________________________________________________________________________________ 
This document is based on 
BM-04-DFC01 
Document Standard for Statistical Analysis Plan (SAP) 
v 15.0 
 
 
 Enterome: EOCRC1-22 
(EN05) 
Statistical Analysis Plan 
 Date: 
06-Mar-2024 
 Version: 
v01 
 Status: 
final 
 Author: 
Vijaya Sankar Kandasamy 
 
Study Title: 
A phase 2 trial of EO2040, a miCrobiaL-derived 
peptide therApeUtic vaccine, in combination with 
nivolumab, for treatment of patients with circulating 
tumor DNA-dEfined minimal residual disease of 
colorectal cancer stage II, III, or IV after completion 
of curative therapy (the "CLAUDE" study). 
Investigational Product: 
EO2040 
Clinical Phase: 
II 
Enrolment of first patient 
19-Jan-2023 (actual date) 
Last Patient Last Visit 
23-Jan-2024 (actual date) 
Sponsor:  
Enterome  
94/96 avenue Ledru-Rollin 75011  
Paris, France 
Project Manager (Sponsor): 
Vincent Arnold 
Data Management and 
Biostatistics (CRO): 
Metronomia Clinical Research GmbH 
Paul-Gerhardt-Allee 42 
81245 München 
Germany 
Project Manager (Metronomia): 
Stefan Schauer (Principal Clinical Data Manager) 
Lead Statistician (Metronomia): 
Katharina Lensing (Principal Statistician) 
Supervisor Biostatistics 
(Metronomia): 
Dorothea Wessiepe (Senior Director, Head of 
Biostatistics) 
 
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Signatures 
 
Authors: 
Statistician, Metronomia Clinical Research GmbH 
Vijaya Sankar Kandasamy  
Date, Signature 
Lead Statistician, Metronomia Clinical Research GmbH 
Katharina Lensing 
Date, Signature 
Approval: 
Head of Data Science, Enterome 
Philippe Rinaudo 
Date, Signature 
Chief Medical Officer, Enterome 
Jan Fagerberg 
Date, Signature 
 
 
07.03.2024 14:36:41 [UTC+1]
Katharina Lensing
08.03.2024 14:57:12 [UTC+1]
Vijaya Sankar Kandasamy
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11-Mar-2024
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Metronomia Clinical Research GmbH 
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This document is based on 
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Document Standard for Statistical Analysis Plan 
(SAP) 
v 15.0 
 
Table of Contents 
Signatures .............................................................................................................................................. 2 
Table of Contents ................................................................................................................................... 3 
1 
Document History .......................................................................................................................... 4 
2 
List of Abbreviations...................................................................................................................... 4 
3 
General ........................................................................................................................................... 5 
3.1 
Analyses Planned and Already Performed ............................................................... 5 
3.2 
SOPs to be Followed ............................................................................................... 5 
4 
Overview of the Protocol ............................................................................................................... 5 
4.1 
Study Objectives ..................................................................................................... 5 
4.1.1 
Primary Objective 
5 
4.1.2 
Secondary Objectives 
5 
4.1.3 
Exploratory Objectives 
6 
4.2 
Study Endpoints ...................................................................................................... 6 
4.2.1 
Primary Endpoint 
6 
4.2.2 
Secondary Endpoint 
7 
4.2.3 
Exploratory Endpoint 
7 
4.3 
Study Design........................................................................................................... 8 
4.4 
Sample Size .......................................................................................................... 10 
4.5 
Study Flow Chart................................................................................................... 10 
5 
General Aspects of the Statistical Analysis ................................................................................ 11 
5.1 
Analysis Sets ........................................................................................................ 11 
5.2 
Protocol Devations ................................................................................................ 11 
5.3 
Intercurrent Events ................................................................................................ 11 
5.4 
Changes or Deviations from Planned Analyses ..................................................... 11 
6 
Definitions for Statistical Analysis .............................................................................................. 11 
6.1 
Baseline and Change from Baseline ...................................................................... 11 
6.2 
Study Day ............................................................................................................. 11 
6.3 
Duration ................................................................................................................ 12 
6.4 
Visit windows and Analysis Visits .......................................................................... 12 
7 
Statistical Analysis Specification ................................................................................................ 12 
7.1 
Specifications Related to Whole Analysis .............................................................. 12 
7.1.1 
Tables 
12 
7.1.2 
Data Listings 
12 
8 
Software and Statistical Programming ....................................................................................... 13 
9 
References ................................................................................................................................... 13 
10 
Changes to Final Version of the SAP .......................................................................................... 14 
11 
List of Tables, Figures and Listings ............................................................................................ 14 
11.1 
Table of Contents for End-of-Text Tables and Figures ........................................... 14 
11.2 
Table of Contents of Individual Patient Data Listings (Appendix 16.2 of the CSR) .. 14 
11.3 
Grading of safety laboratory parameters analogous to NCI CTCAE v5.0 ................ 15 
 
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1 
DOCUMENT HISTORY 
Version 
Date 
Author / editor of new version 
Main changes / comments 
v01 
06-Mar-2024 
Vijaya Sankar Kandasamy 
First version 
2 
LIST OF ABBREVIATIONS 
Abbreviation 
Text 
AE 
Adverse Events 
APS 
All-Patient Set 
ATC 
Anatomical Therapeutic Chemical 
BIRC5 
Baculoviral Inhibitor of Apoptosis Repeat-containing5 
CRC 
Colorectal cancer 
CRF 
Case Report Form 
CSR 
Clinical Study Report 
ctDNA 
Circulating tumor deoxyribonucleic acid 
DFS 
Disease-free survival 
DNA 
Deoxyribonucleic acid 
eCRF 
Electronic Case Report Form 
ELISpot 
Enzyme-linked Immunospot 
FAS 
Full Analysis Set 
FOXM1 
forkheadboxM1 
ICH 
International Council for Harmonization 
IFN-γ 
Interferon-gamma 
IHC 
Immunohistochemistry 
MDSC 
Myeloid-derived suppressor cells 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRD 
Minimal residual disease 
NCI-CTCAE 
National Cancer Institute-Common Terminology Criteria for 
AE 
OS 
Overall Survival 
PD-L1 
Programmed Death-Ligand 1 
PPS 
Per-Protocol Set 
PT 
Preferred Term 
QC 
Quality control 
SAE 
Serious Adverse Event 
SOC 
Standard of Care, System Organ Class 
SOP 
Standard Operating Procedure 
SS 
Safety Set 
TAA 
Tumor associated antigens 
TCR 
T cell Receptor 
TEAE 
Treatment-Emergent Adverse Events 
UCP2 
Universal Cancer Peptide 2 
WHODD 
World Health Organization Drug Dictionary 
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Statistical Analysis Plan 
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3 
GENERAL 
This statistical analysis plan reflects study protocol EOCRC1-22 dated 04-JUL-2022. It 
follows the principles of the Guidelines International Council for Harmonization (ICH) Topic 
E3 and ICH Topic E9. It gives all details for the final statistical analysis of this study.  
3.1 
Analyses Planned and Already Performed 
No formal analysis of the data will be performed as the study is planned to be terminated with 
just one treated patient, only patient data listings will be produced. 
3.2 
SOPs to be Followed 
The analysis will be carried out according to Metronomia Standard Operation Procedures 
(SOPs).  
The report will be written according to the ICH E3 and other applicable ICH guidelines. 
4 
OVERVIEW OF THE PROTOCOL 
4.1 
Study Objectives  
4.1.1 
Primary Objective 
• 
The primary objective of this trial is to assess the 6-month circulating tumor 
deoxyribonucleic acid (ctDNA) clearance rate at therapy with EO2040 in combination 
with nivolumab, in patients with ctDNA-defined minimal residual disease (MRD) of stage 
II-IV colorectal cancer after completion of curative therapy.  
and in sequence, provided the first objective has a positive outcome per protocol 
Section 9:  
• 
The primary objective is, to assess the 6-month ctDNA clearance rate at therapy with 
EO2040 monotherapy, in patients with ctDNA-defined MRD of stage II-IV colorectal 
cancer after completion of curative therapy.  
o In case the first objective does not have a positive outcome per protocol Section 
9, a protocol amendment to adjust the second part of the primary objective to 
utilize an alternative therapeutic vaccine than EO2040 will be considered.   
ctDNA clearance is utilized as a surrogate endpoint for eventual cure, and thereby 
prolongation of disease-free survival (DFS) for patients achieving clearance.   
Note, clearance of ctDNA is characterized by the disappearance of all somatic mutations 
identified in the blood, as well as no appearance of any additional new somatic mutations, 
and radiographic investigation(s) showing no evidence of colorectal cancer (see protocol 
Section 7.8). 
4.1.2 
Secondary Objectives 
To assess the following items at therapy with EO2040 in combination with nivolumab, and 
when applicable EO2040 monotherapy, in patients with ctDNA-defined MRD of stage II-IV 
colorectal cancer after completion of curative therapy:  
• 
Safety and tolerability of study treatments,  
• 
The 3-month ctDNA clearance rate,  
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• 
Progression of colorectal cancer and death as DFS,  
• 
Overall Survival (OS),  
• 
Survival at 36 months after start of study therapy, and  
• 
Induction/expansion of T cells specific for EO2040, the components of EO2040, and 
the targeted nominal tumor associated antigens (TAAs) (Baculoviral Inhibitor of 
Apoptosis Repeat-containing5 [BIRC5] and forkheadboxM1 [FOXM1]). 
4.1.3 
Exploratory Objectives 
• 
To assess expression in tumor tissue (which might be from the primary 
diagnosis/surgery of colorectal cancer (CRC), or a later timepoint before study start; or 
samples from surgeries necessary according to standard of care principles during study 
participation) of TAAs and immune response/resistance related markers.   
• 
To assess shifts in peripheral blood, serum, and tumor tissue (if tissue would also 
become available during study treatment) cell population/marker expressions as for 
instance specific T cell populations, T regulatory cells, myeloid-derived suppressor cells 
(MDSCs), and ctDNA. 
• 
To assess correlations between immunogenicity of EO2040, the components of 
EO2040, and the targeted nominal TAAs (BIRC5 and FOXM1) and efficacy, and safety, 
outcome parameters.  
• 
To characterize T cell receptor (TCR)-variation, and possible correlations with efficacy 
outcome parameters.   
• 
To assess level of tumor mutational burden in individual patients, and possible 
correlations with efficacy outcome parameters.   
• 
To assess disease specific somatic and germline mutations in individual patients related 
to CRC in tumor samples and germline mutations in blood samples, and possible 
correlations with efficacy outcome parameters.  
• 
To assess metagenomics on stool samples to understand the impact of study 
treatments on the microbiome, and possible influence of the microbiome composition of 
patients on safety and efficacy parameters.  
• 
To characterize factors in serum, tumor samples, or blood samples, impacting antitumor 
immunity and definition of individual patient tumor inflammatory signatures, potentially 
delineating patients with a higher likelihood of tumor response to treatment (i.e. 
characterization of factors which may predict clinical benefit).   
• 
To characterize changes in ctDNA allele fractions over time with therapy. 
4.2 
Study Endpoints  
4.2.1 
Primary Endpoint 
• 
The primary endpoint is response to treatment at 6 months (see protocol Section 3.1.1), 
which is defined as having clearance of ctDNA, AND not having any radiographic 
evidence of recurrence at 6 months (see also protocol Section 7.8 for rules of 
assessment of an individual patient best overall response). 
Clearance of ctDNA will be characterized by the disappearance of all somatic mutations 
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identified in the blood, as well as no appearance of any additional new somatic mutations. 
4.2.2 
Secondary Endpoint 
• 
The safety and tolerability of study treatments will be determined by a descriptive 
medical assessment of the combined profile of incidences of adverse events (AEs), 
treatment-emergent AEs (TEAEs), serious AEs (SAEs), deaths, reasons for treatment 
discontinuation/delays, and laboratory abnormalities using the NCI-CTCAE v5.0 grading 
system (see Section 11.3 below and Section 7.7 in protocol).  
• 
Response to therapy at 3 months, defined as clearance of ctDNA, AND no radiographic 
evidence of recurrence (determination method will be the same as for the primary 
endpoint; see protocol Section 3.2.1). 
• 
DFS defined as the time from start of study treatment to the date of first documented 
CRC recurrence or death due to any cause, whichever occurs first. Patients alive without 
recurrence will be censored at the date of last follow-up time. Recurrence is defined by 
the appearance of one or more new CRC lesions on imaging and/or other clinical 
evaluation such as endoscopy.  
• 
OS, measured as the time from start of study treatment until death from any cause. 
Patients alive will be censored at the last time documented to be alive.   
• 
Survival at 36 months after start of study treatment, estimated via Kaplan-Meier 
estimates.  
• 
Immunogenicity and cross reactivity: immunogenicity determined as expansion of 
specific T cells comparing samples taken at baseline versus on treatment in an 
individual patient determining if the patient has a positive response to the immunization, 
or not, in relation to EO2317, EO2318, and Universal Cancer Peptide 2 (UCP2) that 
compose EO2040 by interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot), 
intracellular cytokines staining, and multimers staining assays. Cross reactivities with 
the TAAs BIRC5/survivin and FOXM1 will also be evaluated by the same methods. 
Note, cell availability at each sampling time sets the limit for tests possible to do, and 
the priority order is multimer staining and ELISpot firsthand, and then if possible 
intracellular cytokine staining (the latter might also intentionally not be done in all 
patients). 
4.2.3 
Exploratory Endpoint 
• 
Expression by immunohistochemistry (IHC) in tumor (and normal, if available) tissue of 
BIRC5/survivin, and FOXM1, and immune response/resistance related markers (e.g. 
HLA-A2, Programmed Death-Ligand 1 [PD-L1], CD3, CD4, CD8, granzyme, Ki67; 
additional markers might be added, for instance if immune-resistance mechanisms 
relevant for immunization approaches would become known during trial conduct), 
aiming at defining immune profiles of tumor-infiltrating lymphocytes, and expression of 
immune markers on tumor cells and in the tumor microenvironment via determination of 
percentages of positive cells for the different markers. In addition, and as possible by 
available material, molecular markers (presence, expression levels) will also be 
determined, including but not limited to mutations, deletions, and/or amplifications, and 
necessary assessments for determination of cancer molecular subtypes.  
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• 
Changes between baseline and on treatment evaluations of peripheral blood, serum, 
and tumor tissue cell populations/markers as specific T cell populations (by e.g. TCR 
sequence analyses), T regulatory cells, and MDSCs, changes in immune related 
markers as PD-L1/L2 expression, and changes in ctDNA during the study.  
• 
Correlations between immunogenicity (as measured by specific T cell responses, 
categorizing patients as negative, positive at baseline, or positive during treatment for 
the respective assays) of EO2317, EO2318, and UCP2 and clinical efficacy (per primary 
and secondary efficacy endpoints) and safety (TEAEs of defined specificities and 
grades) outcome parameters. Cross reactivities shown for the TAAs BIRC5/survivin, 
and FOXM1 will also be explored in the same way.  
• 
Characterization of TCR-variation by sequencing, and possible correlations with 
treatment outcomes (per primary and secondary efficacy endpoints).   
• 
Level of tumor mutational burden in individual patients as measured by the total number 
of nonsynonymous mutations per coding area with whole exome sequencing of tumor 
samples, and statistical assessments of correlations of these levels with treatment 
efficacy outcomes (per primary and secondary efficacy endpoints). 
• 
Disease specific somatic and germline mutations in individual patients related to CRC 
as measured by detection of nonsynonymous mutations in tumor samples and germline 
mutations in blood samples by exome sequencing, and correlations with treatment 
efficacy outcomes (per primary and secondary efficacy endpoints).    
• 
Overall fecal microbiome diversity (microbial DNA sequencing for metagenomics) at 
baseline compared with on treatment; changes in relation to safety (TEAEs of defined 
specificities and grades) and efficacy outcome (per primary and secondary efficacy 
endpoints) parameters.   
• 
Characterization of factors impacting antitumor immunity and definition of individual 
patient tumor inflammatory signatures, potentially delineating patients with a higher 
likelihood of tumor response to treatment. These may include measurements in serum 
of cytokines such as IL-6 and inflammatory markers such as C-reactive protein, whole 
exome sequencing of tumors, and single cell RNA-sequencing of PBMCs.  
• 
Characterization of changes in ctDNA allele fractions over time with therapy. 
4.3 
Study Design 
The trial is a multi-center, open-label, non-comparative, two sequential cohort, phase 2 trial, 
to investigate as the first cohort efficacy of the microbiome-derived therapeutic vaccine 
EO2040 in combination with nivolumab in patients with stage II-IV colorectal cancer with 
ctDNA defined MRD after completion of curative therapy. Assuming a positive outcome of 
the first cohort, a second cohort assessing efficacy of EO2040 monotherapy, with the option 
of addition of nivolumab after 3 months in case of no ctDNA clearance, is planned. 
The study is assumed to recruit a total of 17 patients in Cohort 1, and if cohort 1 has a positive 
outcome (see protocol Section 9), a total of 17 patients will also be recruited to Cohort 2A.   
The recruitment period is estimated to be 12 months for Cohort 1, and the same for Cohort 
2A. Thus, in total a possible recruitment time of 24 months. The planned maximum follow-up 
is 36 months for an individual patient, leading to a total study duration of approximately 6 
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years (adding the maximum possible needed 6 months follow-up after recruitment of the last 
patient to Cohort 1 before there is a result to advice on start/no-start of recruitment to Cohort 
2A, and a conservative 6 months for post-last patient visit activities). 
Schematic overview of the trial design, and an overview of the treatment schedule are 
illustrated in figures below. 
Trial design overview: 
 
 
Treatment schedule, scan and ctDNA assessments, and blood sampling for immune 
testing and serum collection: 
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4.4 
Sample Size 
Considering prior attempts in similar situations, the trial will include 17 patients per cohort.  
The primary objective of the trial is to determine the ctDNA clearance rate at 6 months (R6).   
The one-sided null hypothesis H0: R6 ≤ 5% will be tested against the alternative hypothesis 
HA: R6 ≥ 32.5%, using alpha = 0.025 and an exact binomial test. Assuming thus a 
spontaneous ctDNA clearance rate of 5%, and a true ctDNA clearance rate under study 
treatment being at least 32.5% (study treatment being EO2040 in combination with nivolumab 
or EO2040 monotherapy, with the possible addition of nivolumab at 3 months; Cohort 2A) 17 
patients will provide a power of 85%. 
4.5 
Study Flow Chart 
Refer the latest study protocol. 
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5 
GENERAL ASPECTS OF THE STATISTICAL ANALYSIS 
5.1 
Analysis Sets 
The All-Patient (AP) set will consist of any patient who signed informed consent including 
screen-failures.  
The Full Analysis Set (FAS) will consist of patients who received at least one dose of 
EO2040 and for whom no important protocol deviations occurred that would compromise the 
evaluation of efficacy.  
The Safety Set (SS) will consist of patients who received at least one dose of study 
treatment.  
The Per-Protocol (PP) set will consist of patients who received at least 5 doses of EO2040 
(and when applicable associated nivolumab), for whom no important protocol deviations 
occurred, and who have at least one evaluable post-screening ctDNA and tumor imaging.  
5.2 
Protocol Devations 
All protocol deviations regardless of their source will be listed for each patient together with 
the categorization into major or minor.  
5.3 
Intercurrent Events 
Not Applicable.  
5.4 
Changes or Deviations from Planned Analyses 
Since the study is planned to be terminated with just one treated patient, no formal analysis 
of the data will be performed.  
6 
DEFINITIONS FOR STATISTICAL ANALYSIS 
6.1 
Baseline and Change from Baseline  
Baseline is defined as the last non-missing value prior to first administration of study 
treatment. 
Absolute Change from Baseline at a visit/timepoint will be calculated as follows: 
Result at visit/timepoint – Baseline result; 
For assessments taken before and after first administration of study treatment, baseline 
assessments will be flagged separately in the listings. Similarly, change from baseline values 
will be displayed alongside observed results in the respective listings.  
6.2 
Study Day  
The reference day (Day 1) for study day calculations is the date of the first administration 
(V1) of study treatment. The study day will be calculated as follows: 
• 
Assessment/Visit Date – Date of first administration study treatment + 1; for 
assessments on or after the date of first administration of study treatment 
• 
Assessment/Visit Date – Date of first administration study treatment; for assessments 
before the date of first administration of study treatment 
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There is no Study Day 0. 
Study day will be displayed alongside visit/event dates in the relevant listings. 
6.3 
Duration 
Duration of an event will be calculated as follows: 
Duration (days) = (End date of event – Start date of event) +1; 
In case of partial start/end dates, duration will be calculated assuming the 15th of the 
respective months if case of missing day. Duration will not be calculated if months and day 
are not given. 
All listings displaying event data like AE, medication, etc., will include duration of the events 
alongside the end dates. 
6.4 
Visit windows and Analysis Visits 
Data collected by visit will be displayed for the visit it was collected, irrespective of the actual 
date of the assessment. 
The following tables shows the visit labels to be used in patient data listings: 
eCRF visit label 
Visit label in Listing 
Screening (S1) 
Screening 1 
Screening (S2) 
Screening 2 
Visit 1 (W1 – Day 1) 
Week 1 Day 1 
Visit 2 (W3 – Day 15) 
Week 3 Day 15 
Visit 3 (W5 – Day 29) 
Week 5 Day 29 
Visit 4 (W7 – Day 43) 
Week 7 Day 43 
Visit 5 (W11 – Day 71) 
Week 11 Day 71 
Visit 6 (W13 – Day 85) 
Week 13 Day 85 
Visit 7 (W15 – Day 99) 
Week 15 Day 99 
Visit 8 (W19 – Day 127) 
Week 19 Day 127 
Visit 9 (W23 – Day 155) 
Week 23 Day 155 
Visit 10 (W27 – Day 183) 
Week 27 Day 183 
Visit d30 
Safety follow-up 1 
Visit d100 
Safety follow-up 2 
Post-treatment follow-up visits will be displayed as “Survival follow-up 1”, “Survival follow-up 
2”, ... etc. 
7 
STATISTICAL ANALYSIS SPECIFICATION 
7.1 
Specifications Related to Whole Analysis 
7.1.1 
Tables 
No tables will be produced. 
7.1.2 
Data Listings 
All CRF data will be listed as documented. For screen failures only demographic data and 
screen failure details will be listed. Relevant generated variables from section 6 will be listed 
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based on 
BM-04-DFC01 
Document Standard for Statistical Analysis Plan (SAP) 
v 
15.0 
 
next to the original data items as well.  
In all listings, the cohort labels will be displayed as given below:  
• 
Cohort 1  
• 
Cohort 2A 
• 
Cohort 2B 
In listings with screen failure data, screen failures will have cohort label as “Not Applicable” 
and will be sorted at last.  
The listings will be sorted by cohort, patient identifier, visit and date (if applicable).  
Medical history, Procedures and Adverse event: 
All medical history, concomitant illness, procedures and adverse events will be coded using 
MedDRA version mentioned in the latest data management plan and system organ class 
(SOC), preferred term (PT) of the events will be presented in the listing.  
Medication: 
All medication will be coded using World Health Organization Drug Dictionary (WHODD) 
version mentioned in the latest data management plan and Anatomical Therapeutic Chemical 
(ATC) level 2 term, PT of the medications will be presented in the listing. 
8 
SOFTWARE AND STATISTICAL PROGRAMMING 
The statistical analysis will be performed using the SAS® statistical software package in 
Version 9.4.  
SAS programming will be performed according to Metronomia standards as defined in BM-
08-SOP “Statistical Analysis and Programming” and related work instructions. Special 
attention will be paid to planning and performance of quality control measures as documented 
in the QC plan for the analysis of this study (see also BM-08-WIN03 “How to Plan and 
Document QC for Statistical Analysis”). 
9 
REFERENCES 
Common Terminology Criteria for Adverse Events (CTCAE), Version 5.0, Published: 
November 27, 2017. U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, National 
Institutes 
of 
Health 
National 
Cancer 
Institute, 
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/ctcae_v5_quick_r
eference_5x7.pdf. Accessed February 2, 2024. 
ICH Topic E9: Statistical Principles for Clinical Trials, 5 February 1998, adopted by CPMP, 
March 1998, issued as CPMP/ICH/363/96ICH 
ICH E9 (R1): Addendum on Estimands and Sensitivity Analysis in Clinical Trials to the 
Guideline on Statistical Principles for Clinical Trials, 20 November 2019, adopted by CHMP, 
30 January 2020, issued as EMA/CHMP/ICH/436221/2017 
SAS 9.4 https://support.sas.com/documentation/94/index.html. Copyright © SAS Institute 
Inc., SAS Campus Drive, Cary, North Carolina 27513, USA. All rights reserved. 
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This document is 
based on 
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Document Standard for Statistical Analysis Plan (SAP) 
v 
15.0 
 
10 
CHANGES TO FINAL VERSION OF THE SAP 
Not Applicable. 
11 
LIST OF TABLES, FIGURES AND LISTINGS 
11.1 Table of Contents for End-of-Text Tables and Figures 
No tables and figures will be produced. 
11.2 Table of Contents of Individual Patient Data Listings (Appendix 16.2 of the 
CSR) 
16.2. Patient Data Listings 
16.2.1. Discontinued Patients 
16.2.1.1. Patient Enrollment including Reason for Screen Failure – All Patient Set 
16.2.1.2. Study termination including Discontinuation of treatment – Safety Set 
16.2.1.3. Visit Dates – Safety Set 
16.2.2. Protocol Deviations 
16.2.2.1. Protocol Deviations – Safety Set 
16.2.2.2. Violation of Inclusion Criteria – All Patient Set 
16.2.2.3. Violation of Exclusion Criteria – All Patient Set 
16.2.3. Patients Excluded from the Analysis 
16.2.3.1. Analysis Population – All Patient Set 
16.2.4. Demographic Data 
16.2.4.1. Demographics and Baseline Characteristics – All Patient Set 
16.2.4.2. Colorectal Cancer Diagnosis – Safety Set 
16.2.4.3. ctDNA Testing History – Safety Set 
16.2.4.4. Histology and Mutation Status – Safety Set 
16.2.4.5. HLA Testing – Safety Set 
16.2.4.6. Medical History and Concomitant Illness – Safety Set 
16.2.5. Compliance 
16.2.5.1. EO2040 Administration – Safety Set 
16.2.5.2. Nivolumab Administration along with Interruption Details – Safety Set 
16.2.6. Individual Efficacy Response Data  
16.2.6.1. ctDNA Testing – All Patient Set 
16.2.6.2. Cumulative Signatera Variant Data – Safety Set 
16.2.6.3. CT Scans – Safety Set 
16.2.6.4. Survival Status – Safety Set 
16.2.7. Adverse Event Listing (Each Patient) 
16.2.7.1. Adverse Events – Safety Set 
16.2.7.2. Serious Adverse Events – Safety Set 
16.2.7.3. Further Details on Serious Adverse Events – Safety Set 
16.2.7.4. Adverse Events Leading to Treatment Interruption – Safety Set 
16.2.7.5. Adverse Events Leading to Treatment Discontinuation – Safety Set 
16.2.7.6. Death Details – Safety Set 
16.2.7.7. Adverse Events with Changes in CTCAE Grades - Safety Set 
16.2.8. Listing of Individual Laboratory Measurements by Patient 
16.2.8.1. Safety Laboratory 
16.2.8.1.1. 
Hematology incl. Differential Blood Count – Safety Set 
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v 
15.0 
 
16.2.8.1.2. 
Coagulation – Safety Set 
16.2.8.1.3. 
Serum Chemistry – Safety Set 
16.2.8.1.4. 
Urinalysis – Safety Set 
16.2.8.1.5. 
Thyroid Function – Safety Set 
16.2.8.1.6. 
Serum Cortisol – Safety Set 
16.2.8.2. Serum Carcinoembryonic Antigen –Safety Set 
16.2.8.3. Pregnancy Test – Safety Set 
16.2.8.4. Serology – Safety Set 
16.2.8.5. Laboratory Values Outside the Normal Range – Safety Set 
16.2.9. Other Data 
16.2.9.1. Prior and Concomitant Medication – Safety Set 
16.2.9.2. Procedures and Non-drug Therapy – Safety Set Set 
16.2.9.3. Vital Signs and Body Weight – Safety Set 
16.2.9.4. ECOG Performance Status – Safety Set 
16.2.9.5. Physical Examination – Safety Set 
16.2.9.6. Electrocardiogram – Safety Set 
16.2.9.7. Sample Collection for Signatera Assay Setup – Safety Set 
16.2.9.8. Tumor Tissue Sample Collection – Safety Set 
16.2.9.9. Tumor Tissue Collection – Safety Set 
16.2.9.10. PMBC Sample Collection – Safety Set 
16.2.9.11. Serum Sample Collection – Safety Set  
16.2.9.12. Plasma Sample Collection – Safety Set 
16.2.9.13. Stool sample kit dispensation and sample collection – Safety Set 
16.2.9.14. Comments – Safety Set 
16.2.9.15. Unscheduled Investigations – Safety Set 
 
11.3 Grading of safety laboratory parameters analogous to NCI CTCAE v5.0 
Parameter 
Normal 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Hematology 
Hemoglobin (Anemia) 
≥LLN 
<LLN - 10 g/dl
 
 
8 - <10 g/dl 
< 8 g/dl 
- 
Platelets 
≥LLN 
<LLN - 75.0 
x109/L 
50.0 - <75.0 
x109/L 
25.0 - 50.0 
x109/L 
<25.0 x109/L 
White Blood Cells 
(White blood cell 
decreased) 
≥LLN 
<LLN - 3 x109/L 
<3 - 2 x109/L 
<2 - 1 x109/L 
<1 x109/L 
Neutrophils 
decreased 
≥LLN 
<LLN - 1.5 
x109/L 
1.0 - <1.5 
x109/L 
0.5 - <1.0 
x109/L 
<0.5 x109/L 
Lymphocytes 
decreased/increased 
≥LLN and  
≤4.0 x109/L 
<LLN - 0.8 
x109/L  
0.5 - <0.8 
x109/L  
or  
> 4.0 - 20.0 
x109/L 
0.2 - <0.5 
x109/L  
or 
> 20.0 x109/L 
<0.2 x109/L 
Eosinophils 
(Eosinophilia) 
≤ULN 
> ULN and  
> Baseline 
- 
- 
- 
Clinical chemistry 
Alanine 
aminotransferase 
(assuming baseline 
was normal) 
≤ULN 
>ULN - 3.0 x 
ULN  
>3.0 - 5.0 x 
ULN  
>5.0 - 20.0 x 
ULN  
>20.0 x ULN  
Alanine 
aminotransferase 
< 1.5 x baseline 
1.5 - 3.0 x 
baseline 
>3.0 - 5.0 x 
baseline 
>5.0 - 20.0 x 
baseline  
>20.0 x 
baseline  
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 _________________________________________________________________________________________________________________________  
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v 
15.0 
 
Parameter 
Normal 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
(assuming baseline 
was abnormal) 
Aspartate 
aminotransferase 
(assuming baseline 
was normal) 
≤ULN 
>ULN - 3.0 x 
ULN  
>3.0 - 5.0 x 
ULN  
>5.0 - 20.0 x 
ULN  
>20.0 x ULN  
Aspartate 
aminotransferase 
(assuming baseline 
was abnormal) 
< 1.5 x baseline 
1.5 - 3.0 x 
baseline  
>3.0 - 5.0 x 
baseline 
>5.0 - 20.0 x 
baseline 
>20.0 x 
baseline 
Alkaline Phosphatase 
(assuming baseline 
was normal) 
≤ULN 
>ULN - 2.5 x 
ULN  
>2.5 - 5.0 x 
ULN  
>5.0 - 20.0 x 
ULN  
>20.0 x ULN  
Alkaline Phosphatase 
(assuming baseline 
was abnormal) 
< 2.0 x baseline 
2.0 - 2.5 x 
baseline 
>2.5 - 5.0 x 
baseline 
>5.0 - 20.0 x 
baseline 
>20.0 x 
baseline 
Lactate 
dehydrogenase 
≤ULN 
>ULN 
- 
- 
- 
Bilirubin  
(assuming baseline 
was normal) 
≤ULN 
>ULN - 1.5 x 
ULN  
>1.5 - 3.0 x 
ULN  
>3.0 - 10.0 x 
ULN  
>10.0 x ULN  
Bilirubin  
(assuming baseline 
was abnormal) 
≤ baseline 
>1.0 - 1.5 x 
baseline  
>1.5 - 3.0 x 
baseline  
>3.0 - 10.0 x 
baseline 
>10.0 x 
baseline 
Albumin 
(Hypoalbuminemia) 
≥LLN 
<LLN - 30 g/L 
< 30 - 20 g/L 
< 20 g/L 
- 
Sodium  
(Hypernatremia & 
Hyponatremia)  
≤ULN and ≥LLN 
>ULN - 150 
mmol/L or 
<LLN - 130 
mmol/L 
 >150 - 155 
mmol/L or 
<130 - 125 
mmol/L 
>155 - 160 
mmol/L or 
<125 - 120 
mmol/L 
>160 mmol/L or 
<120 mmol/L 
Potassium  
(Hyperkalemia & 
Hypokalemia) 
≤ULN and ≥LLN 
>ULN - 5.5 
mmol/L or 
<LLN - 3.0 
mmol/L 
 >5.5 - 6.0 
mmol/L 
>6.0 - 7.0 
mmol/L  
or 
<3.0 - 2.5 
mmol/L 
>7.0 mmol/L or 
<2.5 mmol/L 
Calcium 
(Hypercalcemia & 
Hypocalcemia) 
≤ULN and ≥LLN 
Corrected 
serum calcium 
of >ULN - 2.9 
mmol/L or  
<LLN - 2.0 
mmol/L 
Corrected 
serum calcium 
of >2.9 - 3.1 
mmol/L or 
<2.0 - 1.75  
mmol/L 
Corrected 
serum calcium 
of >3.1 - 3.4 
mmol/L or  
<1.75 - 1.5  
mmol/L 
Corrected 
serum calcium 
of >3.4 mmol/L 
or 
<1.5 mmol/L 
Creatinine 
≤ULN 
>ULN - 1.5 x 
ULN 
>1.5 - 3.0 x 
ULN  
>3.0 - 6.0 x 
ULN  
>6.0 x ULN  
Glucose 
(Hypoglycemia) 
≥LLN 
<LLN - 3.0 
mmol/L 
<3.0 - 2.2 
mmol/L 
<2.2 - 1.7 
mmol/L 
<1.7 mmol/L 
Coagulation 
Activated Partial 
Thromboplastin Time 
≤ULN 
>ULN - 1.5 x 
ULN  
>1.5 - 2.5 x 
ULN 
>2.5 x ULN 
- 
Prothrombin Time  
(INR increased) 
≤1.2 
>1.2 - 1.5 
>1.5 - 2.5 
>2.5 
 
LLN: Lower limit of normal; ULN: Upper limit of normal; INR: International normalized ratio 
 
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Prot_001.pdf: 
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Version: 2.0 / Date: 2022-JUL-04 
Page 1 on 155  
 
Clinical Trial Protocol EOCRC1-22 
 
 
 
 
 
Investigational Product: 
 
EO2040  
 
EU trial number: 
2022-500664-35-00 
NCT Number 
05350501 
US IND 
28389 
Protocol version number: 
FINAL / version 2.0 
Date: 
2022-JUL-04 
 
 
Property of the Sponsor: 
Enterome 
94/96 avenue Ledru-Rollin 75011 
Paris, France 
 
Sponsor Signatory & Medical Monitor: 
Jan Fagerberg 
Chief Medical Officer, Enterome 
 
 
Enterome Medical Monitor Telephone 
Enterome Medical Monitor E-Mail 
+33 (0) 6 14 31 07 59  
medicalmonitoring-crc@enterome.com  
A phase 2 trial of EO2040, a miCrobiaL-derived peptide 
therApeUtic vaccine, in combination with nivolumab, for treatment 
of patients with circulating tumor DNA-dEfined minimal residual 
disease of colorectal cancer stage II, III, or IV after completion of 
curative therapy 
 (the "CLAUDE" study)   


Protocol No: EOCRC1-22 
CONFIDENTIAL 
 
 
 
Protocol status: FINAL 
Version: 2.0 / Date: 2022-JUL-04 
Page 2 on 155  
CONFIDENTIAL 
This document contains proprietary information & trade secrets of ENTEROME.  
This information may not be used, divulged, published, copied, or otherwise disclosed to any third party without the 
prior written consent of ENTEROME. 
 
 
 


Protocol No: EOCRC1-22 
CONFIDENTIAL 
 
 
 
Protocol status: FINAL 
Version: 2.0 / Date: 2022-JUL-04 
Page 3 on 155  
SIGNATURE FOR PROTOCOL APPROVAL AND RELEASE 
I, the undersigned, have read this protocol and the appendices and agree that they contain all 
the necessary information required for the conduct of the study. 
 
 
 
 
 
 
 
  
Jan Fagerberg 
 
Date 
Chief Medical Officer 
Enterome, Paris, France 
 
 
Sponsor information  
(Achille) CLAUDE Debussy (22 August 1862 – 25 March 1918) was a French composer. 
He is sometimes seen as the first impressionist composer, although he vigorously rejected 
the term. He was among the most influential composers of the late 19th and early 20th 
centuries. 
Born to a family of modest means and little cultural involvement, Debussy showed enough 
musical talent to be admitted at the age of ten to France's leading music college, the 
Conservatoire de Paris. He originally studied the piano, but found his vocation in innovative 
composition, despite the disapproval of the Conservatoire's conservative professors. He took 
many years to develop his mature style and was nearly 40 when he achieved international 
fame in 1902 with the only opera he completed, Pelléas et Mélisande. 
Debussy's 
orchestral 
works 
include Prélude 
à 
l'après-midi 
d'un 
faune (1894), 
Nocturnes (1897–1899) and Images (1905–1912). His music was to a considerable extent a 
reaction against Wagner and the German musical tradition. He regarded the 
classical symphony as obsolete and sought an alternative in his "symphonic sketches", La 
mer (1903–1905). His piano works include sets of 24 Préludes and 12 Études. Throughout 
his career he wrote mélodies based on a wide variety of poetry, including his own. He was 
greatly influenced by the Symbolist poetic movement of the later 19th century. A small 
number of works, including the early La Damoiselle élue and the late Le Martyre de saint 
Sébastien have important parts for chorus. In his final years, he focused on chamber music, 
completing three of six planned sonatas for different combinations of instruments. 
With early influences including Russian and Far Eastern music, Debussy developed his own 
style of harmony and orchestral coloring, derided – and unsuccessfully resisted – by much 
of the musical establishment of the day. His works have strongly influenced a wide range of 
composers including Béla Bartók, Olivier Messiaen, George Benjamin, and the jazz pianist 
and composer Bill Evans. Debussy died from colorectal cancer at his home in Paris at the 
age of 55 after a composing career of a little more than 30 years. 


Protocol No: EOCRC1-22 
CONFIDENTIAL 
 
 
 
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Version: 2.0 / Date: 2022-JUL-04 
Page 4 on 155  
GLOBAL COORDINATING INVESTIGATOR AGREEMENT 
I, the undersigned, have read this protocol and the appendices and agree that they contain 
all the necessary information required for the conduct of the study, and I agree to conduct 
the study as described herein. 
 
 
 
 
 
 
 
 
Doctor Arvind Dasari, MD, MS, 
 
Date 
Associate Professor, Department of  
Gastrointestinal (GI) Medical Oncology,  
Division of Cancer Medicine,  
The University of Texas MD Anderson Cancer Center,  
Houston, TX, USA 
 


Protocol No: EOCRC1-22 
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Page 5 on 155  
EUROPEAN COORDINATING INVESTIGATOR AGREEMENT 
I, the undersigned, have read this protocol and the appendices and agree that they contain 
all the necessary information required for the conduct of the study, and I agree to conduct 
the study as described herein. 
 
 
 
 
 
 
 
 
Prof. Dr. Gunnar Folprecht,  
 
Date 
Department of Oncology,  
University Hospital Carl Gustav Carus,  
Dresden, Germany 
 
 
 
 
 
 
 
 
 
 
 
 


Protocol No: EOCRC1-22 
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Version: 2.0 / Date: 2022-JUL-04 
Page 6 on 155  
SITE PRINCIPAL INVESTIGATOR AGREEMENT 
I, the undersigned, have read this protocol and the appendices and agree to conduct the study 
as described herein. 
 
 
Printed Name:  
 
 
_______________________________________________  
 
 
Site location:  
               
_______________________________________________  
 
Signature: 
 
 
_______________________________________________  
 
 
Date:  
      
_______________________________________________          
 
 
 
 
 
 
 


Protocol No: EOCRC1-22 
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Protocol status: FINAL 
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TABLE OF CONTENT 
LIST OF FIGURES ....................................................................................................................... 11 
LIST OF TABLES ......................................................................................................................... 12 
ABBREVIATIONS ........................................................................................................................ 13 
SYNOPSIS ...................................................................................................................................... 17 
1 
BACKGROUND .......................................................................................................... 32 
1.1 
Colorectal cancer .............................................................................................................. 32 
1.1.1 
Epidemiology, risk factors, and symptoms ............................................................... 32 
1.1.2 
Curative treatment settings ........................................................................................ 33 
1.1.3 
Surveillance after curative treatments ....................................................................... 34 
1.1.4 
Metastatic disease ..................................................................................................... 35 
1.2 
Minimal residual disease setting in colorectal cancer ....................................................... 36 
1.2.1 
Circulating tumor DNA............................................................................................. 36 
1.2.2 
ctDNA-defined minimal residual disease ................................................................. 38 
1.2.3 
Signatera ctDNA assay ............................................................................................. 38 
1.3 
EO2040 a microbiome-derived therapeutic vaccine for treatment of minimal residual 
disease of colorectal cancer ............................................................................................ 39 
1.3.1 
EO2317 and BIRC5/survivin .................................................................................... 41 
1.3.2 
EO2318 and FOXM1 ................................................................................................ 45 
1.3.3 
UCP2 – Helper peptide ............................................................................................. 48 
1.3.4 
Montanide ISA 51 VG .............................................................................................. 50 
1.3.5 
Combination with anti-PD1 blockade ....................................................................... 50 
1.3.6 
Early clinical development of microbiome-derived peptides in solid tumors ........... 51 
1.3.6.1 
EO2401 clinical safety experience ........................................................................ 52 
1.3.6.2 
EO2401 clinical efficacy and immunogenicity ..................................................... 61 
1.3.7 
Benefit/risk assessment and description of and justification for the route of 
administration, dosage, dosage regimen, and treatment period ................................ 63 
1.4 
History of amendments ..................................................................................................... 67 
2 
RATIONALE ................................................................................................................ 68 
2.1 
Rationale for the study ...................................................................................................... 68 
3 
STUDY OBJECTIVES AND ENDPOINTS .............................................................. 69 
3.1 
Objectives ......................................................................................................................... 69 
3.1.1 
Primary objective ...................................................................................................... 69 
3.1.2 
Secondary objectives ................................................................................................. 69 
3.1.3 
Exploratory objectives .............................................................................................. 69 
3.2 
Endpoints .......................................................................................................................... 70 
3.2.1 
Primary endpoint ....................................................................................................... 70 
3.2.2 
Secondary endpoints ................................................................................................. 70 


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3.2.3 
Exploratory endpoints ............................................................................................... 71 
4 
STUDY DESIGN .......................................................................................................... 73 
4.1 
Overall trial design and timelines ..................................................................................... 73 
4.2 
General trial design ........................................................................................................... 75 
4.3 
Trial Safety ....................................................................................................................... 75 
4.4 
End of study ...................................................................................................................... 77 
4.4.1 
Planned study completion and study duration........................................................... 77 
4.4.2 
Early site closure and early study termination .......................................................... 77 
4.5 
Discussion of study design ............................................................................................... 78 
5 
POPULATION ............................................................................................................. 80 
5.1 
Inclusion criteria ............................................................................................................... 80 
5.2 
Exclusion criteria .............................................................................................................. 81 
5.3 
Removal of patients from therapy or study ....................................................................... 84 
5.3.1 
Patient withdrawal of consent ................................................................................... 84 
5.3.2 
Screen failures ........................................................................................................... 84 
5.3.3 
Criteria for treatment discontinuation ....................................................................... 84 
5.3.4 
Lost to follow-up ....................................................................................................... 84 
5.3.5 
Criteria for study participation termination ............................................................... 85 
5.3.6 
Follow-up of patients discontinued from study treatment or withdrawn from study 85 
5.3.7 
Procedures for handling incorrectly enrolled patients ............................................... 85 
5.3.8 
Patient replacement ................................................................................................... 85 
6 
TREATMENT OF PATIENTS ................................................................................... 86 
6.1 
Description of EO2040 ..................................................................................................... 86 
6.1.1 
Pharmaceutical form ................................................................................................. 86 
6.1.2 
Preparation of the emulsion ...................................................................................... 86 
6.1.3 
Packaging and labelling ............................................................................................ 87 
6.1.4 
Storage and stability .................................................................................................. 88 
6.2 
Description of nivolumab ................................................................................................. 88 
6.2.1 
Pharmaceutical form ................................................................................................. 88 
6.2.2 
Preparation of the formulation .................................................................................. 88 
6.2.3 
Packaging and labeling ............................................................................................. 88 
6.2.4 
Storage and stability .................................................................................................. 88 
6.2.5 
Expected safety profile of nivolumab ....................................................................... 89 
6.3 
Administration of EO2040 and the combination of EO2040 and nivolumab ................... 90 
6.4 
Treatment modifications for EO2040 and nivolumab ...................................................... 91 
6.4.1 
Recommended treatment modifications for EO2040 and nivolumab in case of 
immune-related adverse reactions ............................................................................. 92 


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6.5 
Study treatment accountability, reconciliation, and return ............................................... 95 
6.6 
Method of treatment assignment ....................................................................................... 96 
6.7 
Ancillary treatments .......................................................................................................... 96 
6.7.1 
Prior and concomitant treatments and procedures .................................................... 96 
6.7.2 
Permitted concomitant medications .......................................................................... 97 
6.7.3 
Prohibited medications and other therapies .............................................................. 98 
6.7.4 
Contraception ............................................................................................................ 99 
6.8 
Treatment compliance ....................................................................................................... 99 
7 
STUDY ASSESSMENTS AND PROCEDURES ..................................................... 100 
7.1 
Study schedule ................................................................................................................ 100 
7.2 
Screening period #1 ........................................................................................................ 106 
7.3 
Screening period #2 ........................................................................................................ 106 
7.4 
Visits during study treatment .......................................................................................... 109 
7.5 
Post-treatment safety visits ............................................................................................. 111 
7.6 
Visits during post-treatment follow-up ........................................................................... 111 
7.6.1 
Post-treatment visits before determination of relapsed disease ............................... 111 
7.6.2 
Post-treatment visits after determination of relapsed disease ................................. 112 
7.7 
Safety assessments .......................................................................................................... 113 
7.8 
Efficacy assessments....................................................................................................... 113 
7.9 
Primary immunological assessments .............................................................................. 114 
7.9.1 
Cell mediated immunity and associated utilization of PBMCs ............................... 114 
7.9.2 
Correlations between immunogenicity and other trial outcome parameters ........... 115 
7.10 
Assessments for other exploratory endpoints ................................................................. 115 
8 
SAFETY MONITORING .......................................................................................... 116 
8.1 
Safety monitoring definitions ......................................................................................... 116 
8.1.1 
Adverse events ........................................................................................................ 116 
8.1.2 
Serious Adverse events ........................................................................................... 116 
8.1.2.1 
Excluded events .................................................................................................. 116 
8.1.3 
Suspected Unexpected Serious Adverse Reactions (SUSARs) .............................. 117 
8.1.4 
Severity/intensity versus seriousness ...................................................................... 117 
8.2 
Pregnancies ..................................................................................................................... 117 
8.3 
Safety monitoring periods ............................................................................................... 118 
8.3.1 
Reporting ................................................................................................................. 118 
8.3.2 
Follow-up ................................................................................................................ 118 
8.4 
Assessing adverse events ................................................................................................ 119 
8.4.1 
Causality .................................................................................................................. 119 
8.4.2 
Severity/intensity .................................................................................................... 119 


Protocol No: EOCRC1-22 
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8.5 
Reporting by the investigational site .............................................................................. 120 
8.5.1 
Adverse events ........................................................................................................ 120 
8.5.1.1 
Adverse Events Associated with an Overdose or Error in Treatment Administration
 121 
8.5.2 
Documenting in the eCRF ....................................................................................... 121 
8.5.3 
Immediately reportable events - Serious Adverse Events & Pregnancies .............. 122 
8.5.3.1 
SAE minimum notification/reporting requirements ........................................... 123 
8.6 
Independent Data Monitoring Committee (IDMC) ........................................................ 124 
9 
STATISTICAL EVALUATION ............................................................................... 125 
9.1 
Analysis populations ....................................................................................................... 125 
9.2 
Statistical methods .......................................................................................................... 125 
9.3 
Interim analyses .............................................................................................................. 125 
9.4 
Determination of sample size & primary analysis .......................................................... 126 
10 
ADMINISTRATIVE CONSIDERATIONS............................................................. 127 
10.1 
Regulatory and ethical considerations ............................................................................ 127 
10.2 
Finances and insurances .................................................................................................. 127 
10.3 
Informed consent ............................................................................................................ 128 
10.4 
Future use of patient samples; sample traceability ......................................................... 128 
10.5 
Patient data protection .................................................................................................... 128 
10.6 
Site monitoring ............................................................................................................... 129 
10.7 
Data collection ................................................................................................................ 129 
10.8 
Protocol amendments ...................................................................................................... 130 
10.9 
Protocol deviations and violations .................................................................................. 130 
10.10 Change in investigator .................................................................................................... 131 
10.11 Clinical study report........................................................................................................ 131 
10.12 Confidentiality/disclosure ............................................................................................... 131 
10.13 Record retention .............................................................................................................. 131 
10.14 Publications ..................................................................................................................... 132 
11 
LITERATURE ........................................................................................................... 133 
11.1 
References ....................................................................................................................... 133 
12 
APPENDICES ............................................................................................................ 141 
12.1 
Appendix 1: ECOG performance status ......................................................................... 141 
12.2 
Appendix 2: New York Heart Association functional classification .............................. 142 
12.3 
Appendix 3: National Institute of Health (NIH) deviation definitions ........................... 143 
12.4 
Appendix 4: Administration-related reactions ................................................................ 145 
12.5 
Appendix 5: Management algorithms regarding nivolumab for studies under CTCAE 
version 5.0 .................................................................................................................... 147 


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LIST OF FIGURES 
 
Figure 1 : ESMO clinical practice guidelines for localized colon cancer; recommendations for 
follow-up after curative resection. ................................................................................................... 35 
Figure 2 : Microbiome-derived peptide therapeutic vaccine concept .............................................. 40 
Figure 3 : Relative BIRC5 mRNA expression in normal tissues; (HPA RNA-seq normal tissues 
[29]; RPKM: Reads Per Kilobase Million) RNA-seq performed on tissue samples from 95 human 
individuals representing 27 different tissues .................................................................................... 42 
Figure 4 : Kaplan-Meier survival curves for PFS (A) and OS (B) in patients with stage IV 
colorectal cancer (N=71) [44] .......................................................................................................... 43 
Figure 5 : BIRC5 gene expression profile across all tumor samples and paired normal tissues; RNA 
sequencing expression data of 9,736 tumors and 8,587 normal samples from the TCGA and the 
GTEx projects [35] .......................................................................................................................... 44 
Figure 6 : BIRC5 gene expression profile across all colorectal samples and paired normal tissues; 
RNA sequencing expression data of BIRC5 in colon adenocarcinoma (COAD), rectum 
adenocarcinoma tumors (READ) and related normal samples from the TCGA and the GTEx 
projects [35] ..................................................................................................................................... 44 
Figure 7 : Relative FOXM1 mRNA expression in normal tissues; (HPA RNA-seq normal tissues 
[59]; RPKM: Reads Per Kilobase Million) RNA-seq performed on tissue samples from 95 human 
individuals representing 27 different tissues .................................................................................... 46 
Figure 8 : FOXM1 gene expression profile across all tumor samples and paired normal tissues; 
RNA sequencing expression data of 9,736 tumors and 8,587 normal samples from the TCGA and 
the GTEx projects [61] ..................................................................................................................... 47 
Figure 9 : FOXM1 gene expression profile across colorectal samples and paired normal tissues, 
association with overall survival in patients with colon adenocarcinoma; Kaplan–Meier analyses of 
overall survival according to FOXM1 expression levels in adenocarcinoma patients [70] ............. 47 
Figure 10 : TERT gene expression profile across all tumor samples and paired normal tissues; RNA 
sequencing expression data of 9,736 tumors and 8,587 normal samples from the TCGA and the 
GTEx projects [85] ........................................................................................................................... 49 
Figure 11 : Mean tumor growing curves of U87MG glioblastoma engrafted mice following 
adoptive transfer of T cells from HHD DR3 mice vaccinated with EO2315 or human peptide, w/o 
combination with anti-PD1 antibody ............................................................................................... 51 
Figure 12 : EOGBM1-18: Duration of local administration site reactions per clinical cut-off 2021-
OCT-29 based on 38 events reported in 19 patients (of a total of 40 patients) ................................ 57 
Figure 13 : EOGBM1-18: EO2401 induces strong immune response in all patients. Tetramer 
staining after in vitro stimulation, Interpole hyperbola curve from all data points, 95% confidence 
bands. ............................................................................................................................................... 62 
Figure 14 : EOGBM1-18: Cross-reactivity in patients demonstrated for all 3 TAAs (examples). .. 62 
Figure 15 : EOCRC1-22 Overview trial design ............................................................................... 74 
Figure 16 : EOCRC1-22 treatment schedule, scan and ctDNA assessments, and blood sampling for 
immune testing and serum collection ............................................................................................... 74 
 


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LIST OF TABLES 
 
Table 1 : The most common adverse events (cut at 10%), irrespective of relationship noted in trial 
EOGBM1-18 .................................................................................................................................... 54 
Table 2 : The most common treatment-related adverse events (cut at 5%), noted in trial EOGBM1-
18 ...................................................................................................................................................... 55 
Table 3 : The most common (cut at 10% among 43 patients) TEAEs by MedDRA preferred term 
irrespective of relationship in trial EOADR1-19 (Cohorts 2 and 3, combined cohorts) [extracted 
from Table 14.3.3.2.3] ...................................................................................................................... 59 
Table 4 : The most common (cut at 5% among 43 patients) TEAEs related to study treatment by 
MedDRA preferred term in trial EOADR1-19 (Cohorts 2 and 3, combined cohorts) [extracted from 
Table 14.3.3.3.3] .............................................................................................................................. 59 
Table 5 : Composition of the final emulsified EO2040 DP in adjuvant obtained by reconstitution 86 
Table 6 : Nivolumab immune-related adverse reactions leading to permanent discontinuation or 
requiring high-dose corticosteroids .................................................................................................. 90 
Table 7 : Recommended treatment modifications for EO2040 and nivolumab in relation to 
immune-related adverse reactions .................................................................................................... 93 
Table 8 : Study Schedule ................................................................................................................ 101 
Table 9 : Assessment of best overall response ............................................................................... 114 
 
 


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ABBREVIATIONS  
Abbreviation   
Definition 
ACC  
Adrenocortical Carcinoma  
ACTH 
Adrenocorticotropic hormone 
AE 
Adverse Events 
AESI 
Adverse Events of Special Interest 
AIDS 
Acquired Immunodeficiency Syndrome 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
ANC 
Absolute Neutrophil Counts 
Anti-HBcAg 
Antibody to the Hepatitis B Core Antigen 
Anti-HBsAg 
Antibody to the Hepatitis B Surface Antigen 
AP-1 
APC 
Activator Protein 1 
Antigen Presenting Cell  
APTT 
Activated Partial Thromboplastin Time 
ASCO 
American Society of Clinical Oncology 
AST 
Aspartate Aminotransferase 
BIRC5 
Baculoviral Inhibitor of Apoptosis Repeat-containing5 
BOR 
Best Overall Response 
BP 
Blood Pressure 
bpm 
Beats per Minute 
BSA 
Body Surface Area 
BUN 
Blood Urea Nitrogen 
CAPOX  
Capecitabine-oxaliplatin 
CEA 
Carcinoembryonic antigen 
CFR 
Code of Federal Regulations 
CI 
Confidence Interval 
CK 
Creatine Kinase 
CLIA 
Clinical Laboratory Improvement Amendments 
CNS 
Central Nervous System 
COAD 
Colon adenocarcinoma  
COVID 
Corona Virus Disease 
CR 
Complete Response 
CRAs 
Clinical Research Associates 
CRC 
Colorectal cancer 
CRO 
Clinical Research Organisation 
CRP 
C-reactive Protein 
CSR 
Clinical Study Report 
CT 
Computed Tomography  
ctDNA 
Circulating tumor deoxyribonucleic acid 
CTLA-4 
Cytotoxic T-lymphocyte-associated Antigen 4 
DC 
Dendritic Cells  
DFS 
Disease-free survival 
DL 
Dose Level 


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DMSO 
Dimethyl Sulfoxide 
DP 
Drug Product 
EBV 
Epstein Barr Virus 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic Case Report Form 
EDC 
Electronic Data Capture 
ELISA 
Enzyme-linked Immunosorbent Assay 
ELISpot 
Enzyme-linked Immunospot 
ENT 
Ears, Nose, Throat 
EORTC 
European Organisation for Research and Treatment of Cancer 
ESMO 
European Society for Medical Oncology 
FACS 
Fluorescence-activated Cell Sorting 
FAS 
Full Analysis Set 
FD&C 
Food Drug & Cosmetic 
FDG 
FluoroDeoxyGlucose 
FOLFOX  
Leucovorin/5-fluorouracil/oxaliplatin 
FOXM1 
forkheadboxM1 
FPI 
First Patient Included 
FSH 
Follicle-stimulating Hormone 
FVC 
Forced Vital Capacity  
GB 
Glioblastoma 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
GTEX 
Genotype-Tissue Expression 
Hb 
Hemoglobin 
HBsAg 
Hepatitis B Surface Antigen 
HBV 
Hepatitis B virus  
HCV 
Hepatitis C virus 
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigen 
HR 
Heart Rate 
hTERT 
human Telomerase Reverse Transcriptase 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Council for Harmonization 
ICI 
Immune Checkpoint Inhibitor 
IDMC 
Independent Data Monitoring Committee 
IEC 
Independent Ethics Committee 
IFN 
Interferon 
IFN-γ 
Interferon-gamma 
IHC 
Immunohistochemistry 
IL13Rα2 
Interleukin-13 Receptor Alpha-2 
IMP 
Investigational Medicinal Product 


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INR 
International Normalized Ratio 
IR 
Incidence Rates 
IRB 
Institutional Review Board 
ISV 
In Situ Vaccine 
IU 
International Unit 
IUD 
Intra Uterine Device 
IUS 
Intra Uterine hormone-releasing System 
IWRS  
Interactive Web Response System  
LDH 
Lactate Dehydrogenase 
LLN 
Lower Limit of Normal 
LPI 
Last Patient Included 
MDSCs 
Myeloid-derived Suppressor Cells 
MedDRA 
Medical Dictionary for Regulatory Activities 
MESF 
Molecules of Equivalent Soluble Fluorochrome  
MHC 
Major histocompatibility complex 
MMR 
Mismatch repair 
MPP 
Malignant Pheochromocytoma/Paraganglioma 
mRNA 
message Ribonucleic Acid  
MRD 
Minimal residual disease 
MRI 
Magnetic resonance imaging  
MSI 
Microsatellite instability 
NCCN 
National Comprehensive Cancer Network 
NCI-CTCAE 
National Cancer Institute-Common Terminology Criteria for AEs 
NED 
No evidence of disease 
NGS 
Next-generation sequencing 
NSCLC 
Non-Small-Cell Lung Cancer 
NK 
Natural Killer 
OS 
Overall Survival 
PBMC 
PCV 
Peripheral Blood Mononuclear Cells 
Procarbazine, lomustine and vincristine 
PD 
Progressive Disease 
PD1 
Programmed cell Death protein 1 
PDL-1 
Programmed Death-Ligand 1 
PET 
Positron Emission Tomography, 
PFS 
Progression-free Survival 
Ph.Eur. 
European Pharmacopoeia 
PI 
Principal Investigator 
PP 
Per-Protocol 
PS 
Performance Status 
PT 
Preferred Term 
PTT 
Partial Thromboplastin Time 
RANO 
Response Assessment in Neuro-Oncology 
Rb 
Retinoblastoma 
RBC 
Red Blood Cells 


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READ               
Rectum adenocarcinoma 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SARS 
Severe Acute Respiratory Syndrome 
SC 
Subcutaneous 
SEER 
Surveillance, Epidemiology and End Results 
SEM 
Standard error of the mean 
SmPC 
Summary of Product Characteristics 
SOC 
Standard of Care 
SOC 
System Organ Class 
SOCS 
Suppressor of Cytokine Signaling 
SP 
Safety Population 
SUSARs 
TAA 
Suspected Unexpected Serious Adverse Reactions 
Tumor Associated Antigen 
TCR 
T cell Receptor 
TEAE 
Treatment-Emergent Adverse Events 
TERT 
Telomerase Reverse Transcriptase 
TNF 
Tumor Necrosis Factor 
TNM 
TMB 
Tumor Nodes Metastases 
Tumor Mutational Burden 
TRAE 
Treatment-Related Adverse Event 
TSH 
Thyroid-Stimulating Hormone 
UCP2 
Universal Cancer Peptide 2 
ULN 
Upper Limit of Normal 
USP-NF 
United States Pharmacopoeia – National Formulary 
WBCs 
White Blood Cells 
WT 
Wild-type 
 
 
 
 


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SYNOPSIS 
Investigational 
medicinal 
product 
EO2040, is a therapeutic peptide vaccine composed of two microbial-derived peptides 
(EO2317 and EO2318) mimicking cytotoxic T cell (CD8+ T cell) HLA-A2 restricted 
epitopes from two different tumor associated antigens (TAAs), survivin also called 
baculoviral inhibitor of apoptosis repeat-containing protein 5 (BIRC5), and forkhead box 
M1 (FOXM1), respectively. EO2040 also includes the helper peptide (CD4+ T cell 
epitope) universal cancer peptide 2 (UCP2), derived from the human telomerase reverse 
transcriptase catalytic subunit (hTERT).  
The peptide mix EO2040, i.e. drug product (DP), will be emulsified with the adjuvant 
Montanide ISA 51 VG to reach a water in oil emulsion before subcutaneous (SC) 
administration. EO2040 will also be given in combination with the anti-PD1 monoclonal 
antibody nivolumab delivered according to global labels. 
EO2040, the compound to be used in the current trial is a derivative of the compound 
EO2401. EO2040 includes the same components as EO2401, except the microbiome 
derived peptide EO2316, mimicking an epitope on the TAA interleukin 13 receptor 
alpha-2 (IL-13Rα2). The reason to exclude peptide EO2316 from compound EO2040 is 
that IL-13Rα2 currently does not have a rationale to be used when colorectal cancer is 
targeted, i.e. expression levels as known today are not high enough.  
Note, the parent compound of EO2040, i.e. EO2401, in combination with nivolumab, 
has been investigated in two phase 1/2 trials in more than 100 patients with glioblastoma 
and adrenal tumors, showing the ability to generate strong systemic immune responses 
against the targeted TAAs, and being well tolerated; the safety profile being consistent 
with the profile of the combination partner nivolumab, with the only addition of local 
administration site reactions (most commonly grade 1, erythema, induration, and 
sometimes pain).  
Protocol 
number 
EOCRC1-22 
Title 
A phase 2 trial of EO2040, a miCrobiaL-derived peptide therApeUtic vaccine, in 
combination with nivolumab, for treatment of patients with circulating tumor DNA-
dEfined minimal residual disease of colorectal cancer stage II, III, or IV after completion 
of curative therapy (the "CLAUDE" study).   
Background  
and rationale 
Colorectal cancer (CRC) is the third most common tumor in men and the second in 
women, accounting for 10% of all tumor types worldwide. Incidence is 25% higher in 
males and differs greatly between countries. With more than 600,000 deaths estimated 
each year, CRC is the fourth most common cancer-related cause of death globally. 
In 2020, an estimated 104,610 new cases of colon cancer and 43,340 cases of rectal 
cancer was to occur in the USA. During the same year, an estimated 53,200 people will 
die of colon and rectal cancer combined. The mortality rate in the European Union is 15-
20 out of 100,000 in males and 9-14 out of 100,000 in females and has decreased over 
time, particularly in females. In affected European individuals, 5-year survival ranges 
from 28.5% to 57% in men and from 30.9% to 60% in women, with a pooled estimation 
in 23 countries of 46.8% in men and 48.4% in women. 
After appropriate diagnostic measures surgical intervention is the first key step in 
treatment of colon cancer. Assuming surgery for cure, i.e. R0-resection with 
microscopically margin-negative resection and no gross or microscopic tumor remaining 
in the primary tumor bed, the assessment of risk of recurrence is important in deciding 
when to recommend systemic adjuvant treatment with the aim of reducing risk of relapse 
and death. The risk of relapse after colon cancer resection is estimated by integrating the 
clinicopathological features of the tumor with the molecular marker mismatch repair 
(MMR)/microsatellite instability (MSI) status.  
TNM staging remains the most relevant histological criteria for risk assessment after 
surgery of colon cancer. Reported 5-year survival rates after surgical resection alone are 
99% for stage I, 68%-83% for stage II and 45%-65% for stage III disease.  


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In general, it has been established that adjuvant systemic therapy decreases the risk of 
death by an absolute 3%-5% in high-risk stage II colon cancer with single-agent  
5-fluorouracil (5-FU) and by 10%-15% in stage III disease with fluoropyrimidines alone, 
with a further 4%-5% improvement with oxaliplatin-containing combinations. 
FOLFOX (leucovorin/5-fluorouracil/oxaliplatin) and CAPOX (capecitabine plus 
oxaliplatin) remain the current standard of care adjuvant treatments for stage III disease. 
Note, irinotecan, cetuximab and bevacizumab have not demonstrated clinical activity in 
the localized disease setting and therefore they should never be used as adjuvant 
treatment in this setting.  
Studies of selected patients undergoing surgery to remove CRC liver metastases (i.e. 
stage IV) have shown that cure is possible in this population and should be the goal for 
a substantial number of these patients. Reports have shown 5-year disease-free survival 
(DFS) rates of approximately 20% in patients who have undergone resection of liver 
metastases, and a recent meta-analysis reported a median 5-year survival of 38%.  
Despite all efforts regarding surgery and adjuvant therapy per above, approximately 25% 
of patients who present with localized disease will later develop metastases. In addition, 
of new colorectal cancer diagnoses, 20% of patients have metastatic disease already at 
presentation.  
Metastatic disease not amenable to local treatment only (e.g. surgery, stereotactic body 
radiation therapy, haptic arterial infusion, arterial embolization therapy, tumor ablation 
via for instance radiofrequency ablation or cryotherapy and other means), is usually 
treated with systemic chemotherapy or targeted treatments as outlined in different 
guidance documents.  
In 
addition 
to 
fluoropyrimidine-, 
oxaliplatin-, 
and/or 
irinotecan-containing 
chemotherapy regimens, immunotherapy and targeted therapy regimens are becoming an 
increasingly important part of the treatment landscape for patients with metastatic CRC. 
Combination of a biologic agent (e.g. bevacizumab, cetuximab, panitumumab) with 
some of the chemotherapy regimens is an option, depending on available data. Systemic 
therapy options for patients with progressive disease depend on the choice of initial 
therapy and biomarker status of the tumor. 
As outlined in the ESMO guidance regarding metastatic CRC, a patient with classical 
metastatic CRC may typically achieve an OS of approximately 30 months as the result 
of a multidisciplinary team-managed ‘continuum of care’. Among people diagnosed with 
metastatic colorectal cancer, approximately 70%-75% of patients survive beyond 1 year, 
30%-35% beyond 3 years, and fewer than 20% beyond 5 years from diagnosis. 
Thus, CRC continues to be a major therapeutic challenge with a considerable number 
of patients experiencing premature death from early disease recurrence. Historically, 
it has been challenging to definitively identify patients with CRC at risk of recurrence 
after completed assumed curative treatment, which has complicated the ability to 
optimize adjuvant drug development. Relapsed CRC may occur in the absence of 
symptoms, further emphasizing the importance of sensitive screening modalities. 
Circulating tumor DNA (ctDNA) can be detected at varying concentrations across 
multiple cancer types and disease stages. ctDNA typically constitutes a small proportion 
of an individual’s total circulating cell-free DNA (cfDNA), <1% according to some 
studies. However, with improving assay techniques providing greater levels of 
sensitivity, the analysis of ctDNA is rapidly being accepted as a reliable tool in oncology.  
For patients with early-stage solid tumors, ctDNA testing may identify patients who have 
residual disease or recurrence after potentially curative treatments (surgery in particular). 
Based on this evidence, multiple clinical trials have been initiated to evaluate the clinical 
validity and utility of this approach.  
An analysis from the large GALAXY study, an observational study monitoring minimal 
residual disease, MRD, evaluating the association of ctDNA dynamics with a short-term 
clinical outcome and adjuvant chemotherapy efficacy was recently presented. A total 
1,365 patients with CRC were enrolled between June 2020 and April 2021 and included 
in the analysis; 116 stage I, 478 stage II, 503 stage III, and 268 oligometastatic resectable 


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stage IV. The analysis demonstrated the association of ctDNA dynamics with improved 
clinical outcomes in MRD+ patients; also showing that stratifying post-surgical 
treatment decisions using a ctDNA assay can identify patients likely to benefit from 
adjuvant chemotherapy across all stages, including stage II.  
In contrast to the analysis of tumor biopsy samples, which are not only invasive to 
obtain but often also do not fully capture tumor heterogeneity and evolution, the 
analysis of ctDNA offers a non-invasive method of longitudinally evaluating the 
genomic profile of a patient’s cancer. Building on, among others, the above referenced 
study, data on the potential uses of ctDNA are rapidly accumulating in the continuum 
of care across multiple cancers.  
ctDNA is composed of short fragments of tumor-derived DNA (roughly 130-150 base 
pairs) that are released by cancer cells into the circulation via apoptosis, necrosis, or 
secretion.  Detectable ctDNA after completion of curative therapies has nearly 100% 
positive predictive value for radiographic recurrence. Multiple studies demonstrate that 
the presence of ctDNA likely reflects radiographically unseen micrometastases, or MRD, 
which is predictive of recurrence and a useful screening tool for high-risk patients.  
The lead-time of ctDNA positivity to radiographic recurrence is approximately 9 months 
in early-stage disease, which provides a window for evaluation of novel therapies in a 
setting for which there are no current standard-of-care guidelines.  
Clearance of ctDNA may be used as an endpoint in early-stage studies to stratify 
patients for more definitive trials and promote drug development in this space. The 
improved detection of ctDNA has provided a unique opportunity to use a noninvasive 
approach to explore therapeutic options and to potentially eradicate micrometastatic 
disease in high-risk patients.  
Extensive pre-clinical studies have been conducted to characterize the microbiome-
derived mimicry therapeutic vaccine concept as outlined in the figure below, and two 
multi-peptide compounds have been evaluated in clinical trials (EO2401 in glioblastoma, 
adrenocortical carcinoma, and malignant pheochromocytoma/paraganglioma; EO2463 
in follicular and marginal zone lymphoma). 
 
EO2040, the microbiome-derived mimicry peptide mix for treatment of CRC, has been 
developed to maximize the ability to generate a strong immune response, by high affinity 
MHC (Major histocompatibility complex)  class I binding-peptides (i.e. targeting 
presentation for CD8+ cytotoxic T cells), high capacity to be recognized by the immune 
system by its non-self-nature, and possibility for fast expansion of T cells by the pre-
existence of T cell clones already recognizing the mimicry peptides which can be 
efficiently reactivated by a vaccine boost leading to a durable immune response. The 
concomitant administration of EO2040 and an anti-PD1 blocking agent is utilized to 
optimize the possibility for tumor specific T cells to infiltrate and effectively function 
within the tumor environment. 


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The two tumor targeting peptides in EO2040, i.e. EO2317 and EO2318, were found in 
the human microbiota, and are restricted to HLA-A2 expressed in 49% of the Caucasian 
population. The microbiome-derived peptides display high sequence homologies with 
two different TAAs: survivin also called baculoviral inhibitor of apoptosis repeat-
containing protein 5 (BIRC5), and forkhead box M1 (FOXM1), respectively. These 
TAAs are highly expressed in CRC, but not in normal tissues. The two different TAAs 
were selected to overcome possible tumor heterogeneity and reduce tumor escape. The 
peptides demonstrated in in vitro and in vivo pre-clinical testing high MHC binding 
affinity, and ability to elicit strong immune responses as well as cross reactivity against 
the human corresponding peptides.  
The microbiome-derived therapeutic vaccine concept utilized in conjunction with anti-
PD1 blockade is an innovative option for testing of a rational immunotherapy in CRC. 
The concept as such, including the combination with nivolumab, has already been 
tested in the clinical setting (i.e. in recurrent glioblastoma and adrenal tumors) and 
shown to be well tolerated, capable of expanding tumor target specific T cells very 
rapidly and extensively, and having the ability to shrink advanced solid tumors.   
Based on the above, the current study will evaluate the microbiome-derived therapeutic 
vaccine EO2040 in combination with nivolumab in patients with circulating tumor 
DNA-defined MRD of colorectal cancer stage II, III, or IV after completion of 
standard curative therapy. 


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Objectives 
Primary objective  
 
The primary objective of this trial is to assess the 6-month ctDNA clearance rate at 
therapy with EO2040 in combination with nivolumab, in patients with ctDNA-
defined MRD of stage II-IV colorectal cancer after completion of curative therapy. 
and in sequence, provided the first objective has a positive outcome (see Primary 
Endpoint): 
 
The primary objective is, to assess the 6-month ctDNA clearance rate at therapy with 
EO2040 monotherapy, in patients with ctDNA-defined MRD of stage II-IV 
colorectal cancer after completion of curative therapy. 
ctDNA clearance is utilized as a surrogate endpoint for eventual cure, and thereby 
prolongation of disease-free survival (DFS) for patients achieving clearance.  
Note, clearance of ctDNA is characterized by the disappearance of all somatic mutations 
identified in the blood, as well as no appearance of any additional new somatic 
mutations, and radiographic investigation(s) showing no evidence of colorectal cancer. 
Secondary objectives 
To assess the following: 
 
safety and tolerability of study treatments, 
 
the 3-month ctDNA clearance rate, 
 
progression of colorectal cancer and death as disease-free survival (DFS), 
 
overall survival (OS), 
 
survival at 36 months after start of study therapy, and 
 
induction/expansion of T cells specific for EO2040, the components of EO2040, and 
the targeted nominal TAAs (BIRC5 and FOXM1).  
Exploratory objectives 
1. To assess expression in tumor tissue (which might be from the primary 
diagnosis/surgery of CRC, or a later timepoint before study start; or samples from 
surgeries necessary according to standard of care principles during study 
participation) of TAAs and immune response/resistance related markers.  
2. To assess shifts in peripheral blood, serum, and tumor tissue (if tissue would also 
become available during study treatment) cell population/marker expressions as for 
instance specific T cell populations, T regulatory cells, myeloid-derived suppressor 
cells (MDSCs), and ctDNA. 
3. To assess correlations between immunogenicity of EO2040, the components of 
EO2040, and the targeted nominal TAAs (BIRC5 and FOXM1) and efficacy, and 
safety, outcome parameters. 
4. To characterize T cell receptor (TCR)-variation, and possible correlations with 
efficacy outcome parameters.  
5. To assess level of tumor mutational burden in individual patients, and possible 
correlations with efficacy outcome parameters.  
6. To assess disease specific somatic and germline mutations in individual patients 
related to CRC in tumor samples and germline mutations in blood samples, and 
possible correlations with efficacy outcome parameters. 
7. To assess metagenomics on stool samples to understand the impact of study 
treatments on the microbiome, and possible influence of the microbiome 
composition of patients on safety and efficacy parameters. 
8. To characterize factors in serum, tumor samples, or blood samples, impacting 
antitumor immunity and definition of individual patient tumor inflammatory 
signatures, potentially delineating patients with a higher likelihood of tumor 
response to treatment (i.e. characterization of factors which may predict clinical 
benefit).  
9. To characterize changes in ctDNA allele fractions over time with therapy.  


Protocol No: EOCRC1-22 
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Endpoints 
 
Primary endpoint 
Response to therapy will be measured with a standard-of-care, CLIA certified ctDNA 
assay, or similar certification outside of the USA (timepoint 6 months).  
Identification of somatic mutations in the blood will be confirmed by sequencing of the 
resected tumor using a CLIA-certified next-generation sequencing panel. Clearance of 
ctDNA will be characterized by the disappearance of all somatic mutations identified in 
the blood, as well as no appearance of any additional new somatic mutations.  
The primary endpoint is response to treatment at 6 months, which is defined as having 
clearance of ctDNA, AND not having any radiographic evidence of recurrence at 6 
months.  
If at least 4 out of 17 patients in the Full Analysis Set (FAS; patients who received at 
least one dose of EO2040 and for whom no important protocol deviations occurred) show 
response to study therapy, the therapy will be declared successful. 
Secondary endpoints 
1. The safety and tolerability of study treatments will be determined by a descriptive 
medical assessment of the combined profile of incidences of adverse events (AEs), 
treatment-emergent AEs (TEAEs), serious AEs (SAEs), deaths, reasons for 
treatment discontinuation/delays, and laboratory abnormalities using the NCI-
CTCAE v5.0 grading system. 
2. Response to therapy at 3 months, defined as clearance of ctDNA, AND no 
radiographic evidence of recurrence (determination method will be the same as for 
the primary endpoints).   
3. Disease-free survival (DFS) defined as the time from start of study treatment to the 
date of first documented CRC recurrence or death due to any cause, whichever 
occurs first. Patients alive without recurrence will be censored at the date of last 
follow-up time. Recurrence is defined by the appearance of one or more new CRC 
lesions on imaging and/or other clinical evaluation such as endoscopy. 
4. Overall survival (OS), measured as the time from start of study treatment until death 
from any cause. Patients alive will be censored at the last time documented to be 
alive.  
5. Survival at 36 months after start of study treatment, estimated via Kaplan-Meier 
estimates. 
6. Immunogenicity and cross reactivity: Immunogenicity determined as expansion of 
specific T cells comparing samples taken at baseline versus on treatment in an 
individual patient determining if the patient has a positive response to the 
immunization, or not, in relation to EO2317, EO2318, and UCP2 that compose 
EO2040 by interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot), 
intracellular cytokines staining, and multimers staining assays. Cross reactivities 
with the TAAs BIRC5/survivin and FOXM1 will also be evaluated by the same 
methods. Note, cell availability at each sampling time sets the limit for tests possible 
to do, and the priority order is multimer staining and ELISpot firsthand, and then if 
possible intracellular cytokine staining (the latter might also intentionally not be 
done in all patients).  
Exploratory endpoints 
1. Expression by immunohistochemistry (IHC) in tumor (and normal, if available) 
tissue of BIRC5/survivin, and FOXM1, and immune response/resistance related 
markers (e.g. HLA-A2, PD-L1, CD3, CD4, CD8, granzyme, Ki67; additional 
markers might be added, for instance if immune-resistance mechanisms relevant for 
immunization approaches would become known during trial conduct), aiming at 
defining immune profiles of tumor-infiltrating lymphocytes, and expression of 
immune markers on tumor cells and in the tumor microenvironment via 
determination of percentages of positive cells for the different markers. In addition, 
and as possible by available material, molecular markers (presence, expression 
levels) will also be determined, including but not limited to mutations, deletions, 


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and/or amplifications, and necessary assessments for determination of cancer 
molecular subtypes. 
2. Changes between baseline and on treatment evaluations of peripheral blood, serum, 
and tumor tissue cell populations/markers as specific T cell populations (by e.g. TCR 
sequence analyses), T regulatory cells, and myeloid-derived suppressor cells 
(MDSCs), changes in immune related markers as PD-L1/L2 expression, and changes 
in ctDNA during the study. 
3. Correlations between immunogenicity (as measured by specific T cell responses, 
categorizing patients as negative, positive at baseline, or positive during treatment 
for the respective assays) of EO2317, EO2318, and UCP2 and clinical efficacy (per 
primary and secondary efficacy endpoints) and safety (TEAEs of defined 
specificities and grades) outcome parameters. Cross reactivities shown for the TAAs 
BIRC5/survivin, and FOXM1 will also be explored in the same way. 
4. Characterization of T cell receptor (TCR)-variation by sequencing, and possible 
correlations with treatment outcomes (per primary and secondary efficacy 
endpoints).  
5. Level of tumor mutational burden in individual patients as measured by the total 
number of nonsynonymous mutations per coding area with whole exome sequencing 
of tumor samples, and statistical assessments of correlations of these levels with 
treatment efficacy outcomes (per primary and secondary efficacy endpoints).   
6. Disease specific somatic and germline mutations in individual patients related to 
CRC as measured by detection of nonsynonymous mutations in tumor samples and 
germline mutations in blood samples by exome sequencing, and correlations with 
treatment efficacy outcomes (per primary and secondary efficacy endpoints).   
7. Overall fecal microbiome diversity (microbial DNA sequencing for metagenomics) 
at baseline compared with on treatment; changes in relation to safety (TEAEs of 
defined specificities and grades) and efficacy outcome (per primary and secondary 
efficacy endpoints) parameters.  
8. Characterization of factors impacting antitumor immunity and definition of 
individual patient tumor inflammatory signatures, potentially delineating patients 
with a higher likelihood of tumor response to treatment. These may include 
measurements in serum of cytokines such as IL-6 and inflammatory markers such 
as C-reactive protein, whole exome sequencing of tumors, and single cell RNA-
sequencing of PBMCs. 
9. Characterization of changes in ctDNA allele fractions over time with therapy. 
Design 
The trial is a multi-center, open-label, non-comparative, two sequential cohort, phase 2 
trial, to investigate as the first cohort efficacy of the microbiome-derived therapeutic 
vaccine EO2040 in combination with nivolumab in patients with stage II-IV colorectal 
cancer with ctDNA defined MRD after completion of curative therapy. Assuming a 
positive outcome of the first cohort (see Primary Endpoint), a second cohort assessing 
efficacy of EO2040 monotherapy, with the option of addition of nivolumab after 3 
months in case of no ctDNA clearance, is planned.  
The study is assumed to recruit a total of 17 patients in Cohort 1, and if Cohort 1 has a 
positive outcome, a total of 17 patients will also be recruited to Cohort 2A. Thus, 
provided a positive outcome of Cohort 1, a total of 34 patients are assumed to start study 
treatment.   
Generally, no replacement is foreseen. However, the primary analysis for each cohort 
builds on an assessment of 17 patients in the Full Analysis Set (FAS; patients who 
received at least one dose of EO2040 and for whom no important protocol deviations 
occurred). Thus, if there would be relevant important protocol deviation(s) in a patient 
impacting the possibility to assess the primary endpoint the patient might be replaced 
based on a decision by the Sponsor after consultation with the IDMC.  
The recruitment period is estimated to be 12 months for Cohort 1, and the same for 
Cohort 2A. Thus, in total a possible recruitment time of 24 months. The planned 
maximum follow-up is 36 months for an individual patient, leading to a total study 


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duration of approximately 6 years (adding the maximum possible needed 6 months 
follow-up after recruitment of the last patient to Cohort 1 before there is a result to advice 
on start/no-start of recruitment to Cohort 2A, and a conservative 6 months for post-last 
patient visit activities).   
Overview trial design 
 
 
EOCRC1-22 treatment schedule, scan and ctDNA assessments, and 
blood sampling for immune testing and serum collection  
 
There will be a 2-stage consent procedure for the trial, including a first consent for basic 
disease, age information, and HLA-typing (or the use of already available information in 
the patients' file; such information even if based on testing before the screening period is 


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acceptable) since a large proportion of the patients (approximately half of the patients) 
will not match the HLA-A2 prerequisite to receive the HLA-A2 specific EO2040 
immunization compound.  
The second stage of the consent procedure will be related to treatment, and follow-up 
aspects of the trial procedures.  
Patients who have provided informed consent per above, and who meet all the inclusion 
criteria, and do not meet any of the exclusion criteria, can be finally evaluated by the 
Sponsors Medical Monitor for start study treatment. The recruiting site should submit a 
start of treatment request (after also having filled out all applicable information in the 
eCRF) to the Medical Monitor at least 24 hours before anticipated study treatment start 
(procedures are described in a separate Cohort Administration Charter); the Medical 
Monitor will assess the request within 12 hours of receipt.   
Each patient will individually participate in the study for a maximum of approximately 
3.5 years, including approximately 1 month for screening, 6 months for study treatment, 
and a maximum of 3 years follow-up after start of study treatment. 
Study treatments will be administered until study treatment termination criteria as 
outlined in Section 5.3.3 are fulfilled, e.g. reaching maximum study treatment period (6 
months), unacceptable toxicity, or relapsing disease. At the time of stopping study 
treatment, appropriate other treatment will be advised by the treating physician on an 
individual patient basis. The patient should continue, even if study treatment is stopped, 
study follow-up measures for as long as the individual patient consent for follow-up is 
not withdrawn, the site is open, and the study not terminated per plan, or early, but for a 
maximum length of 3 years after start of study treatment. 
All assessments of possible relapse in the trial will be determined at the sites by the 
principal investigators, or designees, according to the standard clinical criteria used at 
the site. 
Of note, there is no current knowledge publicly available regarding the potential of 
pseudoprogression at immunotherapy in patients with ctDNA defined MRD of CRC. 
Thus, at scanning, especially at the 3-month study timepoint, if a relapse is indicated 
by scanning it is recommended to, if at all possible, also document the relapse with 
histology or cytology.  
Visit schedule  
Section 7 of the trial protocol outlines study assessments and procedures, and their 
timings (including visit windows) in the Study Schedule. 
Inclusion/ 
exclusion 
criteria 
Inclusion criteria 
To be eligible to receive study treatment, a patient must meet all the criteria below: 
1. Provided written informed consent prior to any study-related procedures (initial 
consent is for screening part #1 procedures, and a second consent is for screening 
part #2 procedures; see Section 7.2 and Section 7.3, respectively). 
2. Histological confirmation of colorectal cancer (confirmation at initial diagnosis is 
sufficient). 
3. Post R0-resection (i.e. microscopically margin-negative resection and no gross or 
microscopic tumor remaining in the primary tumor bed) of stages II, III, or IV CRC 
and completion of all planned standard of care perioperative and/or adjuvant 
therapies. 
a. Note, if there is doubt the patient has received all relevant standard of care 
therapy, the patient should be discussed with the trial Medical Monitor, 
who can also defer the case for decision by the Global Coordinating 
Investigator. 
4. Presence of minimal residual disease as defined by a positive ctDNA assay after 
completion of all planned standard of care therapies. 
a. Note, patients may be identified for enrollment with any CLIA-certified, or 
similar certification outside of the USA, ctDNA assay for MRD as standard 


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of care or in a surveillance trial. However, MRD status will be confirmed 
with the Signatera assay prior to initiation of study therapy (i.e. within the 
screening part #2 procedures), for all patients who have not had a 
Signatera research grade testing where all parameters from the testing can 
be provided for the current trial. 
5. Age ≥ 18 years old. 
6. Human leukocyte antigen (HLA)-A2 positive. 
7. No evidence of radiographic disease (computer tomography or magnetic resonance 
imaging; optimized to detect metastatic disease, e.g. with contrast as applicable) that 
requires immediate therapeutic intervention as assessed by the treating physician, 
within 28 days of (before or after) a positive ctDNA assay.  
a. Note, if there are lesions on scanning which are highly suspicious for being 
metastatic CRC even if no immediate therapeutic intervention is needed, 
this is a clear indication to NOT enroll the patient in the trial. At doubt the 
case should be discussed with the trial Medical Monitor, who can also defer 
the case for decision by the Global Coordinating Investigator. 
b. Note, if the scanning is not done within -7 days/+28 days of the positive 
Signatera assay which is accepted as confirmatory test for the trial, the 
scanning needs to be repeated within the screening part #2 procedures. 
8. ECOG performance status 0 or 1 (see Section 12.1).  
9. Female patients of childbearing potential must have a negative serum pregnancy test 
within 72 hours prior to start of study therapy. 
10. Considering the embryofetal toxicity of the immune checkpoint inhibitor (ICI) 
shown in animals’ models, the following recommendations for contraception must 
be followed: 
a. 
If not surgically sterile, female patients of childbearing potential age must 
use highly effective contraception from signing the Informed Consent 
Form (ICF) through 6 months after the last treatment dose administered. 
Highly effective contraception includes (according to Clinical Trial 
Facilitation Group: Recommendations related to contraception and 
pregnancy testing in clinical trials [104]): 
i. combined (estrogen and progesterone containing) hormonal 
contraception associated with inhibition of ovulation: oral, 
intravaginal, transdermal, 
ii. progestogen-only hormonal contraception associated with 
inhibition of ovulation: oral, injectable, implantable,   
iii. intrauterine device (IUD), 
iv. intrauterine hormone-releasing system (IUS), 
v. bilateral tubal occlusion, 
vi. vasectomized partner, and 
vii. sexual abstinence when in line with the preferred and usual 
lifestyle of the patient (e.g. periodic abstinence is not considered 
a highly effective method). 
In each case of delayed menstrual period (over 1 month between menstruations), 
confirmation of absence of pregnancy is strongly recommended. This 
recommendation also applies to women of childbearing potential with infrequent or 
irregular menstrual cycles. 


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b. If not surgically sterile, male with female partner of childbearing potential 
age must use condom from signing the ICF through 6 months after the last 
treatment dose administered. Males must ensure that their partners of 
childbearing potential use highly effective contraception also. Note, the 
contraception guidelines for males are not related to nivolumab but kept 
as a conservative precaution in this early development protocol in relation 
to EO2040. 
11. Patients willing and able to comply with the scheduled visits, treatment plan, 
laboratory tests, and other study procedures indicated in the protocol. 
Exclusion criteria 
Patients who meet any of the following criteria will not be eligible to participate in the 
study: 
1. Patients treated with dexamethasone > 2 mg/day or equivalent (i.e., 13 mg/day of 
prednisone) within 14 days before start of study therapy, unless required to treat an 
adverse event (AE). 
Note: The criterion implies the patient should not receive treatment with 
dexamethasone > 2 mg/day or equivalent at the actual time of a screening visit 
(single time point assessment), and within 14 days before the start of study treatment 
(unless required to treat an AE); the latter part of the criterion should be checked at 
the time of start of treatment. 
2. Patients treated with radiotherapy within 12 weeks, and cytotoxic chemotherapy 
therapy within 28 days (or 5 half-lives of the compound(s) administered if longer) 
before study treatment start. 
3. Patients with persistent Grade ≥ 2 toxicities (according to NCI-CTCAE v5.0). 
Toxicities must be resolved for at least 2 weeks to Grade 1 or less. However, 
alopecia, neuropathy, and other persisting toxicities not constituting a safety risk 
based on Investigator’s judgment are acceptable. 
4. Patients who have received any prior treatment with compounds targeting PD1, PD-
L1, CTLA-4, or similar compounds where general resistance against therapeutic 
vaccination approaches might have developed.  
5. Patients with the following abnormal laboratory values: 
a. 
Hemoglobin < 10 g/dL (6.2 mmol/L); transfusion is acceptable to reach the 
value. 
b. Absolute neutrophil count decrease (<1.5 x109/L).  
c. 
Platelet count decrease (< 75 ×109/L). 
d. Total bilirubin > 1.5 ×upper limit of normal (ULN; according to the 
performing laboratory’s reference ranges); except participants with Gilbert 
Syndrome who must have a total bilirubin level of < 3.0 xULN.  
e. 
Alanine aminotransferase (ALT) > 3 ×ULN. 
f. 
Aspartate aminotransferase (AST) > 3 ×ULN. 
g. Serum creatinine increase (> 1.5 ×ULN). 
h. Abnormal thyroid function per local laboratory levels; note, patients with 
hypothyroidism only requiring hormone replacement therapy, and patients 
with long term (judged by the treating physician) stable antithyroid therapy 
due to hyperthyroidism, are permitted to enroll. Patients with abnormal 
thyroid laboratory values judged by the treating physician as clinically non-
relevant are also eligible. 


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6. Patients with presence of other concomitant active, invasive malignancies that may 
interfere with ctDNA analysis (known clonal hematopoiesis of unknown potential 
allowed). 
7. Patients with clinically significant active infection, cardiac disease, significant 
medical or psychiatric disease/condition that, in the opinion of the Investigator, 
would interfere with the interpretation of patient safety or study results or that would 
prohibit the understanding or rendering of informed consent (i.e. only consent able 
patients can be enrolled in the study) and compliance with the requirements of the 
protocol – including (but not limited to): 
a. 
Bacterial sepsis, COVID-19, or other similarly severe infections (clinical 
assessment is the basis for exclusion of severe infections; if clinical 
suspicion, adequate testing should be performed to exclude severe 
infections). 
b. New York Heart Association > Grade 2 congestive heart failure within 
6 months prior to start of study therapy (see Section 12.2). 
c. 
Uncontrolled or significant cardiovascular disease, including: 
i. myocardial infarction within 6 months prior to start of study 
therapy, 
ii. uncontrolled/unstable angina within 6 months prior to start of 
study therapy, 
iii. diagnosed or suspected congenital long QT syndrome, and 
iv. any history of clinically significant ventricular arrhythmias (such 
as ventricular tachycardia, ventricular fibrillation, or Torsades de 
pointes). 
d. Stroke within 6 months prior to start of study therapy. 
e. 
Concurrent neurodegenerative disease. 
f. 
Dementia or significantly altered mental status. 
8. Patients with suspected autoimmune or active autoimmune disorder or known 
history of an autoimmune neurologic condition (e.g., Guillain-Barré syndrome). 
Note-1, patients with a diagnosed inflammatory bowel disease as ulcerative colitis 
or Crohn’s disease, should be excluded. 
Note-2, patients with vitiligo, type I diabetes mellitus, hypothyroidism due to 
autoimmune condition only requiring hormone replacement therapy, psoriasis not 
requiring systemic therapy, or conditions not expected to recur in the absence of an 
external trigger are permitted to enroll. 
9. Patients with a history of solid organ transplantation or allogeneic hematopoietic 
stem cell transplantation. 
10. Patients with a history or known presence of tuberculosis. 
11. Pregnant and breastfeeding patients. 
12. Patients with a history or presence of human immunodeficiency virus (HIV) and/or 
active hepatitis B virus (HBV)/hepatitis C virus (HCV). 
13. Patients who have received live or attenuated vaccine therapy used for prevention of 
infectious diseases including seasonal (influenza) vaccinations within 4 weeks of the 
first dose of study drug. 
14. Patients with a history of hypersensitivity to any excipient, or active substance, 
present in the pharmaceutical forms of applicable study treatments. 


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15. Patients under treatment with immunostimulatory or immunosuppressive 
medications, including herbal remedies, or herbal remedies known to potentially 
interfere with major organ function. 
16. Patients who have received treatment with any other investigational agent, or 
participation in another clinical trial (clinical trial including active interventions; 
participation in clinical trials for data collection purposes only are permitted) within 
28 days (or 5 half-lives if longer) prior to start of study therapy. Note, during 
participation in the current study, parallel participation in other interventional 
studies is not allowed (participation in clinical trials for data collection purposes 
only are permitted; also, in the follow-up parts of interventional studies).   
Investigational 
and standard 
of care 
treatments 
Investigational treatments 
EO2040, is a therapeutic peptide vaccine composed of two microbial-derived peptides 
mimicking cytotoxic T cell (CD8+ T cell) epitopes from the TAAs BIRC5/survivin and 
FOXM1, combined with the helper peptide (CD4+ T cell epitope) UCP2.  
The peptide mix EO2040, i.e. drug product (DP), will be emulsified with the adjuvant 
Montanide ISA 51 VG to reach a water in oil emulsion before subcutaneous (SC) 
administration.  
EO2040 will be given in combination with nivolumab, which is an anti-PD1 fully human 
monoclonal antibody (immunoglobulin G4), blocking the interaction between PD1 and 
its ligands (PD-L1 and PD-L2), in another group of patients EO2040 will also be 
evaluated as monotherapy.  
Nivolumab is approved for use for the treatment of multiple cancer types, including 
subtypes of CRC (mismatch repair deficient or microsatellite instability-high metastatic 
disease after prior treatment). However, it is not currently approved for ctDNA defined 
MRD of CRC. Treatment with nivolumab will follow the current version of European 
SmPC and US PI. 
General standard of care treatments  
All non-cancer therapies that the Investigator feels appropriate are allowed in this study, 
except for the medications outlined in protocol Section 6.7.3. Thus, patients should 
receive full supportive care during participation in the trial and standard of care treatment 
per local practice and according to the judgment of the Investigator or treating physician. 
The use of systemic corticosteroids and other immunosuppressants (beside trial 
treatment) should be avoided because of their potential interference with the 
pharmacodynamic activity of the investigational treatment. However, the use of systemic 
corticosteroids and other immunosuppressants might be of vital importance for patient 
safety in relation to treatment of adverse reactions. Further guidance regarding steroid 
treatment can be found in the trial protocol Section 6.7.2.  
Independent 
Data 
Monitoring 
Committee 
(IDMC) 
The IDMC will serve as an external monitoring group for the study. The primary role of 
the IDMC will be to examine the safety and tolerability of study participants throughout 
the duration of the study. The IDMC will be created to further protect the rights, safety, 
and well-being of patients who will be participating in the trial by monitoring their 
progress and results.  
The IDMC will examine the safety data on an ongoing basis regarding any related Grade 
≥ 4 events, and regarding related Grade 3 events if deemed necessary by the investigator 
responsible for the patient in question or Sponsor. In addition, a pre-planned safety 
assessment will take place at the end of Cohort 1. 
In relation to the efficacy assessment for Cohort 1 (and linked possible transition to 
Cohort 2A), the IDMC will also oversee and validate the efficacy assessment as 
performed by the Sponsor (or designee). 
The IDMC will be consulted by the Sponsor in relation to possible replacement of 
patients in FAS (see Study Design). 
The IDMC members, including a minimum of three qualified persons, are to be 
international hematology/oncology experts, or qualified clinical trial statistician (there is 


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no demand for statistical expertise among the IDMC members for this trial), who are not 
in other ways than via the IDMC involved in the current trial. Details of the IDMC 
processes and procedures is outlined in a separate IDMC Charter. 
Statistical 
considerations 
Statistical methods 
All data collected in this trial will be reported by patient data listings, summary tables, 
and figures for all demographic and baseline characteristics, medical history, efficacy, 
and safety variables. Proportions and the denominators will be provided to summarize 
response, toxicity, and other categorical variables.  
The response rate R6 will be estimated using all patients in the FAS with response at 6 
months in the numerator and all patients in the FAS in the denominator; and the analysis 
will be repeated based on all patients in the PP population. Two-sided exact 95% 
confidence intervals will be provided. A treatment is defined to be successful, if the one-
sided null hypothesis of R6 being smaller or equal to 5% (H0: P ≤ 5%) can be rejected 
on a one-sided 2.5% significance level. DFS and overall survival will be analyzed using 
the method of Kaplan and Meier and presented with two-sided 95% confidence bands. 
Statistical assessments of correlations between e.g. immunogenicity parameters and 
efficacy and safety outcome parameters will be conducted as described in the Statistical 
Analysis Plan (SAP).  
The SAP will provide all details with respect to patients being analyzed for all endpoints. 
Relevant subgroups based on available data and compliance to protocol procedures might 
be defined. 
Statistical tests may be planned in the SAP or performed as needed. The importance of 
the tests does not reside in making conclusions on statistical relevance of differences, but 
rather identification of possible results that could be worth further exploration. 
Determination of sample size & primary analysis  
This is an early development, open-label, exploratory, trial to include patients with CRC 
who have early disease which today is without an established standard of care, but still 
in a relevantly early stage of the overall disease history that there is a potential chance to 
significantly delay recurrence/progression with an efficacious therapy. Thus, a balance 
needs to be struck between a size to find a relevant signal, and the importance to not 
expose too many patients to a possibly non-efficacious therapy.  
Considering prior attempts in similar situations, the trial will include 17 patients per 
cohort. Utilizing FAS as the population for the primary analysis, the basic assumption is 
that there will be no non-evaluable patients. However, if there would be any doubt if a 
patient would be included in the FAS, or not, the Sponsor will consult the IDMC and if 
recommended the patient might be replaced. 
A conservative estimate is that the accrual rate will be approximately 1.5 patients per 
month considering the outlined inclusion/exclusion criteria (see Section 5.1 and Section 
5.2). 
The primary objective of the trial is to determine the ctDNA clearance rate at 6 months 
(R6).  
The one-sided null hypothesis H0: R6 ≤ 5% will be tested against the alternative 
hypothesis HA: R6 ≥ 32.5%, using alpha = 0.025 and an exact binomial test. Assuming 
thus a spontaneous ctDNA clearance rate of 5%, and a true ctDNA clearance rate under 
study treatment being at least 32.5% (study treatment being EO2040 in combination with 
nivolumab or EO2040 monotherapy, with the possible addition of nivolumab at 3 
months; Cohort 2A) 17 patients will provide a power of 85%.  
For the primary analysis, the ctDNA clearance rate will be estimated along with the 95% 
exact confidence interval. The efficacy of study treatment will also be assessed by 
performing a binomial test comparing the ctDNA clearance rate against the null 
hypothesis 5%, at a one-sided alpha level of 0.025.  


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If at least 4 out of 17 patients in the Full Analysis Set (FAS; patients who received at 
least one dose of EO2040 and for whom no important protocol deviations occurred) show 
response to study therapy in a cohort, the therapy will be declared successful. 
Assuming 4 of the 17 patients would achieve a response, the observed response rate 
would be 24% and the corresponding 95% exact confidence interval would be (7.1% to 
50.4%).   
The opening of a Cohort 2A exploring EO2040 monotherapy (with the option of addition 
of nivolumab if ctDNA is not cleared at 3 months) is dependent on the outcome of Cohort 
1; the main factor being the assessment of ctDNA clearance rate (which should be 
positive per above), but also DFS might be considered. If no continuation of exploration 
of EO2040 would be seen as appropriate based on data from Cohort 1, there will be 
considerations regarding a protocol amendment to switch EO2040 to an alternative 
microbiome-derived vaccine.  
Based on the above, it is assumed that the current trial will include enough patients to 
provide safety and tolerability information, immunogenicity data, as well as preliminary 
efficacy data in the selected treatment setting without demanding a too high (and long) 
patient recruitment and without exposing too many patients. 
Interim 
analyses 
The nature of the study, i.e. early exploratory development trial aiming at generating as 
much knowledge as possible before potential decisions related to further development of 
EO2040, makes it important to assess especially safety, but also efficacy and possible 
biomarkers, on an ongoing basis during trial conduct. Exploratory preliminary data from 
the trial might be utilized in relation to e.g. scientific discussions and presentations to 
facilitate input regarding possible improvements of development parameters. 
A definitive interim analysis will be performed at the end of Cohort 1 regarding the 
primary objective, secondary objectives, and exploratory objectives. The timing of the 
analysis is to be linked to the time when the last patient to be analyzed (among the 
patients in Cohort 1 who has not relapsed at an earlier timepoint) has had his/her week 
27 examinations. 
Number of 
patients, sites, 
location, and 
predicted 
durations 
Number of patients  
 
The study is to recruit 34 patients (17 patients in each of two sequential cohorts). 
Locations and number of sites  
 
USA, 2-3 sites, and Germany, 3-5 sites 
Recruitment time, max study therapy time, and follow-up time  
 
Recruitment time approximately 12 months for each of two sequential cohorts. 
 
Max study therapy time 6 months. 
 
Each patient will participate in the treatment and follow-up of the study for 
approximately 36 months. 
Total study duration is expected to be 6 years, including a total recruitment time of 24 
months, a maximum follow-up of 36 months for an individual patient, adding the 
maximum possible needed 6 months follow-up after recruitment of the last patient to 
Cohort 1 before there is a result to advice on start/no-start of recruitment to Cohort 2A, 
and a conservative 6 months for post-last patient visit activities.   
 
 


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1 
BACKGROUND 
1.1 
Colorectal cancer 
1.1.1 
Epidemiology, risk factors, and symptoms 
Colorectal cancer (CRC) is the third most common tumor in men and the second in women, 
accounting for 10% of all tumor types worldwide. Incidence is 25% higher in males and 
differs greatly between countries. With more than 600 000 deaths estimated each year, CRC 
is the fourth most common cancer-related cause of death globally [11]. 
In 2020, an estimated 104,610 new cases of colon cancer and 43,340 cases of rectal cancer 
will occur in the USA [2]. During the same year, an estimated 53,200 people will die of 
colon and rectal cancer combined. Despite these high numbers, the incidence of colon and 
rectal cancers per 100,000 people decreased from 60.5 in 1976 to 46.4 in 2005, and more 
recently, 38.7 in 2016 2].  
The mortality rate in the European Union is 15-20 out of 100,000 in males and 9-14 out of 
100,000 in females and has decreased over time, particularly in females. In affected 
European individuals, 5-year survival ranges from 28.5% to 57% in men and from 30.9% to 
60% in women, with a pooled estimation in 23 countries of 46.8% in men and 48.4% in 
women [11]. 
The risk of developing colon cancer depends on factors which can be classified into lifestyle 
or behavioral characteristics and genetically determined factors. Age is considered the major 
unchangeable risk factor for sporadic colon cancer: nearly 70% of patients are >65 years of 
age and this disease is rare before the age of 40 years, even though data from Western 
registries show an increased incidence in the 40- to 44-year age group 1Individuals with any 
of the following are considered at high risk of colon cancer and must be actively screened 
and in case of inherited syndromes, also referred for genetic counselling: 
 a medical history of adenoma, colon cancer, inflammatory bowel disease (Crohn’s 
disease and ulcerative colitis),  
 significant family history of CRC or adenoma,  
 an inherited cancer syndrome (2%-5% of all CRC), such as familial adenomatous 
polyposis coli and its variants (1%), Lynch-associated syndromes (hereditary non-
polyposis colon cancer) (2%-4%), Turcot, Peutze-Jeghers, and MUTYH-associated 
polyposis syndrome. 
Colon cancer arises from the mucosa of the bowel, growing both into the lumen and the 
bowel wall, and/or spreading to adjacent organs. Symptoms are associated with relatively 
large tumors and/or advanced disease stages and may not be specific for colon cancer. 
Alterations in bowel habit, general or localized abdominal pain, weight loss without other 
specific causes, weakness, iron deficiency and anemia are the most common symptoms and 
depend on the location and stage of the primary tumor [2].  
 


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1.1.2 
Curative treatment settings 
After appropriate diagnostic measures surgical intervention is the first key step in treatment 
of colon cancer [1, 2].  
Assuming surgery for cure, i.e. R0-resection with microscopically margin-negative resection 
and no gross or microscopic tumor remaining in the primary tumor bed, the assessment of 
risk of recurrence is important in deciding when to recommend systemic adjuvant treatment 
with the aim of reducing risk of relapse and death. The risk of relapse after colon cancer 
resection is estimated by integrating the clinicopathological features of the tumor with the 
molecular marker mismatch repair (MMR)/microsatellite instability (MSI) status [1].  
TNM staging remains the most relevant histological criteria for risk assessment after surgery 
of colon cancer. Reported 5-year survival rates after surgical resection alone are 99% for 
stage I, 68%-83% for stage II and 45%-65% for stage III disease [1]. In addition, for 
intermediate stage II, further parameters need consideration to refine the evaluation of risk 
given the observed variability on prognosis [1]. 
In general, it has been established that adjuvant systemic therapy decreases the risk of death 
by an absolute 3%-5% in high-risk stage II colon cancer with single-agent 5-fluorouracil (5-
FU) and by 10%-15% in stage III disease with fluoropyrimidines alone, with a further 4%-
5% improvement with oxaliplatin-containing combinations [1]. 
FOLFOX 
(leucovorin/5-fluorouracil/oxaliplatin) 
and 
CAPOX 
(capecitabine 
plus 
oxaliplatin) remain the current standard of care adjuvant treatments for stage III disease. 
Note, irinotecan, cetuximab and bevacizumab have not demonstrated clinical activity in the 
localized disease setting and therefore they should never be used as adjuvant treatment in 
this setting [1]. Learned societies gives further guidance on treatments to use, or not to use, 
in specific stages of disease and length of treatments which are preferable from a benefit/risk 
perspective [1, 2, 5].  
Studies of selected patients undergoing surgery to remove CRC liver metastases have shown 
that cure is possible in this population and should be the goal for a substantial number of 
these patients [2]. Reports have shown 5-year disease-free survival (DFS) rates of 
approximately 20% in patients who have undergone resection of liver metastases, and a 
recent meta-analysis reported a median 5-year survival of 38% [2]. In addition, retrospective 
analyses and meta-analyses have shown that patients with solitary liver metastases have a 5-
year overall survival (OS) rate as high as 71% following resection [2].  
 
 


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1.1.3 
Surveillance after curative treatments 
The NCCN panel recommendation for post-treatment surveillance for patients with stage 
II/III disease who have undergone successful treatment (i.e. no known residual disease) 
include the following procedures [2]: 
 History and physical examination should be given every 3 to 6 months for 2 years, 
and then every 6 months for a total of 5 years. 
 A carcinoembryonic antigen (CEA) test is recommended at baseline and every 3 to 
6 months for 2 years, then every 6 months for a total of 5 years for patients with stage 
III disease and those with stage II disease if the clinician determines that the patient 
is a potential candidate for aggressive curative surgery. 
 Colonoscopy is recommended at approximately 1 year after resection (or at 3-6 
months post-resection if not performed preoperatively because of an obstructing 
lesion). Repeat colonoscopy is typically recommended at 3 years, and then every 5 
years thereafter, unless follow-up colonoscopy indicates advanced adenoma, in 
which case colonoscopy should be repeated in 1 year. 
 Chest, abdominal, and pelvic CT scan are recommended every 6 to 12 months for up 
to 5 years in patients with stage III disease and those with stage II disease at a high 
risk of recurrence. 
The NCCN panel recommendation for surveillance of patients with stage IV CRC with no 
evidence of disease (NED) after curative-intent surgery and subsequent adjuvant treatment 
are similar to those listed for patients with stage II/III disease, except that certain evaluation 
are performed more frequently. The NCCN panel recommends that these patients undergo 
contrast-enhanced CT scan of the chest, abdomen, and pelvis every 3 to 6 months in the first 
2 years after adjuvant treatment and then every 6 to 12 months for up to a total of 5 years 
[2]. CEA testing is recommended every 3 to 6 months for the first 2 years and then every 6 
months for a total of 5 years, as for patients with earlier stage disease [2]. 
Patients with an elevated CEA level after resection should be investigated with chest, 
abdominal and pelvic CT scan, and physical examination, and consideration of a PET/CT-
scan. If imaging study results are normal in the face of a rising CEA, repeat CT scans are 
recommended every 3 months until either disease is identified, or CEA level stabilizes or 
declines [2]. 
The ESMO guideline regarding localized colon cancer is in principle similar to the NCCN 
panel recommendations for stage II/III disease (Figure 1 [1]). However, the ESMO 
guidelines does not give more specific recommendations regarding surveillance after 
potentially curative surgery of stage IV disease, or at an increase of CEA during surveillance 
[1, 3].    
 
 


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Figure 1 : ESMO clinical practice guidelines for localized colon cancer; 
recommendations for follow-up after curative resection. 
 
 
CEA, carcinoembryonic antigen; CT, computed tomography; mCRC, metastatic colorectal cancer. 
 
1.1.4 
Metastatic disease  
Despite all efforts regarding surgery and adjuvant therapy per above, approximately 25% of 
patients who present with localized disease will later develop metastases [3]. In addition, of 
new colorectal cancer diagnoses, 20% of patients have metastatic disease already at 
presentation [3].  
The above numbers are consistent with the estimates presented by NCCN, that 
approximately 50%-60% of patients diagnosed with CRC develop metastatic disease, and 
80%-90% of these patients have unresectable metastatic liver disease [2]. Also, that 
metastatic disease most frequently develops metachronously after treatment for locoregional 
CRC, with the liver being the most common site of involvement, and that 20%-34% of 
patients with CRC present with synchronous liver metastases [2].  
Metastatic disease not amenable to local treatment only (e.g. surgery, stereotactic body 
radiation therapy, haptic arterial infusion, arterial embolization therapy, tumor ablation via 
for instance radiofrequency ablation or cryotherapy and other means), is usually treated with 
systemic chemotherapy or targeted treatments as outlined in different guidance documents 
[1, 2].  
According to the NCCN guideline, recommendations for patients with disseminated 
metastatic disease represent a continuum of care in which lines of treatment are blurred rather 
than discrete [2]. In addition to fluoropyrimidine-, oxaliplatin-, and/or irinotecan-containing 
chemotherapy regimens, immunotherapy and targeted therapy regimens are becoming an 
increasingly important part of the treatment landscape for patients with metastatic CRC. 
Combination of a biologic agent (e.g. bevacizumab, cetuximab, panitumumab) with some of 
the chemotherapy regimens is an option, depending on available data. Systemic therapy 


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options for patients with progressive disease depend on the choice of initial therapy and 
biomarker status of the tumor. 
As outlined in the ESMO guidance regarding metastatic CRC [4], a patient with classical 
metastatic CRC may typically achieve an OS of approximately 30 months as the result of a 
multidisciplinary team-managed ‘continuum of care’. An example of a typical ‘continuum 
of care’ treatment sequence is outlined below: 
 approximately 4–6 months of first-line ‘induction’ therapy, 
 4–6 (–8) months of ‘maintenance’ therapy - or no treatment after resection and/or 
ablation following first-line treatment, 
 about 3 months re-introduction (or treatment beyond progression) 
 5–7 months of second-line therapy, 
 a treatment break before initiation of a further line, 
 approximately 3 months of third-line therapy, 
 potentially a fourth line (in patients with RAS wild-type disease),  
 a few months of re-challenge of initial induction or first-line therapy, and 
 a few months best supportive care only. 
Among people diagnosed with metastatic colorectal cancer, approximately 70%-75% of 
patients survive beyond 1 year, 30%-35% beyond 3 years, and fewer than 20% beyond 5 
years from diagnosis [3]. 
Thus, CRC continues to be a major therapeutic challenge with a considerable number of 
patients experiencing premature death from early disease recurrence. Historically, it has 
been challenging to definitively identify patients with CRC at risk of recurrence after 
completed assumed curative treatment, which has complicated the ability to optimize 
adjuvant drug development. Relapsed CRC may occur in the absence of symptoms, further 
emphasizing the importance of sensitive screening modalities. 
1.2 
Minimal residual disease setting in colorectal cancer 
Wan et al. has published a thorough review regarding liquid biopsies for residual disease 
and recurrence, covering the background necessary for current trial [6]. 
1.2.1 
Circulating tumor DNA 
Circulating fragments of cell-free DNA were first described in 1948 by Mandel and Métais 
[7]. Proof-of-principle studies demonstrated the potential clinical utility of detecting 
circulating tumor DNA (ctDNA) in the 1990s. For example, DNA fragments carrying 
mutations in the KRAS gene were identified in the plasma of patients with pancreatic cancer 
using allele-specific polymerase chain reaction (PCR) [8]. Multiple studies, using sensitive 
mutation analysis in plasma of cancer patients and xenograft models, confirmed that ctDNA 
fragments are derived from cancer cells and could be used as a quantitative marker to assess 
cancer disease burden [9, 10, 11]. In parallel, the introduction of massively parallel or next-
generation sequencing (NGS) has enabled an astonishing progress in cancer genomics from 
both tissue and body fluid samples [12]. 


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ctDNA can be detected at varying concentrations across multiple cancer types and disease 
stages [13]. ctDNA typically constitutes a small proportion of an individual’s total 
circulating cell-free DNA, <1% according to some studies. However, with improving assay 
techniques providing greater levels of sensitivity, the analysis of ctDNA is rapidly being 
accepted as a reliable tool in oncology. The proportion of patients in whom ctDNA can be 
detected depends on the extent of tumor volume. For instance, in patients with CRC, the 
proportion of patients with detectable ctDNA ranges from 50% in those with non-metastatic 
disease to nearly 90% in patients with metastatic disease. 
In patients with advanced-stage disease, where levels of ctDNA are higher, detection of 
genomic alterations in specific genes by analysis of plasma samples can be used to guide 
targeted therapies, and analysis of ctDNA can be used to assess for instance overall mutation 
rates (potential to inform on the likelihood of efficacy of immunotherapies), and response to 
treatment or identify progression (fractional concentration monitored over time) [6].  
An important example of the use of personalized ctDNA analysis as a predictive biomarker 
in patients with solid tumors treated with immune checkpoint blockade (pembrolizumab) 
was recently published by Bratman et al [14]. The study investigators applied ctDNA assays 
to 316 serial plasma samples obtained at baseline and every three cycles from 94 patients; 
baseline ctDNA concentration correlated with progression-free survival, overall survival, 
clinical response and clinical benefit. This association became stronger when considering 
ctDNA kinetics during treatment. Of note, all 12 patients with ctDNA clearance during 
treatment were alive with median 25 months follow up. According to the authors "This study 
demonstrates the potential for broad clinical utility of ctDNA-based surveillance in patients 
treated with immune checkpoint blockade".  
For patients with early-stage solid tumors, ctDNA testing may identify patients who have 
residual disease or recurrence after potentially curative treatments (surgery in particular) [15, 
16, 17, 18, 19]. On the basis of this evidence, multiple clinical trials have been initiated to 
evaluate the clinical validity and utility of this approach [6].  
An analysis from the large GALAXY study, an observational study monitoring MRD, 
evaluating the association of ctDNA dynamics with a short-term clinical outcome and 
adjuvant chemotherapy efficacy was recently presented [23]. A total 1,365 patients with 
CRC were enrolled between June 2020 and April 2021 and included in the analysis; 116 
stage I, 478 stage II, 503 stage III, and 268 oligometastatic resectable stage IV. The analysis 
demonstrated the association of ctDNA dynamics with improved clinical outcomes in 
MRD+ patients; also showing that stratifying post-surgical treatment decisions using a 
ctDNA assay can identify patients likely to benefit from adjuvant chemotherapy across all 
stages, including stage II [23].  
In contrast to the analysis of tumor biopsy samples, which are not only invasive to obtain 
but often also do not fully capture tumor heterogeneity and evolution, the analysis of 
ctDNA offers a non-invasive method of longitudinally evaluating the genomic profile of a 
patient’s cancer. Building on, among others, the above referenced studies, data on the 


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potential uses of ctDNA are rapidly accumulating in the continuum of care across multiple 
cancers.  
1.2.2 
ctDNA-defined minimal residual disease 
ctDNA is composed of short fragments of tumor-derived DNA (roughly 130-150 base pairs) 
that are released by cancer cells into the circulation via apoptosis, necrosis, or secretion [20].  
Detectable ctDNA after completion of curative therapies has nearly 100% positive predictive 
value for radiographic recurrence. Multiple studies demonstrate that the presence of ctDNA 
likely reflects radiographically unseen micrometastases, or minimal residual disease (MRD), 
which is predictive of recurrence and a useful screening tool for high-risk patients [21].  
The lead-time of ctDNA positivity to radiographic recurrence is up to 9 months in early-
stage disease, which provides a window for evaluation of novel therapies in a setting for 
which there are no current standard-of-care guidelines [22].  
Clearance of ctDNA may be used as an endpoint in early-stage studies to stratify patients 
for more definitive trials and promote drug development in this space. The improved 
detection of ctDNA has provided a unique opportunity to use a noninvasive approach in 
order to explore therapeutic options and to potentially eradicate micrometastatic disease 
in high-risk patients.  
1.2.3 
Signatera ctDNA assay 
The Signatera (Natera) MRD blood test is custom-built for each patient, to track 16 single-
nucleotide variants that were previously detected in the patient’s tumor, but not in the 
patient’s normal DNA (clonal mutations), and likely to be present in all tumor cells 
regardless of tumor evolution. To call a test positive, at least 2 out of 16 single-nucleotide 
variants must be observed above the confidence threshold. A test result consists of a 
qualitative call on presence/absence of ctDNA, as well as precise ctDNA quantification in 
terms of mean tumor molecules per mL of plasma. In the clinical setting, the Signatera 
assay will have a turnaround time of 1 week, provided the tumor normal whole-exome 
sequencing analysis has been completed, which will require 10-14 calendar days. 
Analytical studies have shown over 90% sensitivity at 0.01% variant allele frequency, with 
analytical specificity >99.8%. In clinical validation studies, recurrence hazard ratios of 
17.5 and 43.5, were demonstrated based on the results of a single test and serial testing 
after definitive treatment, respectively. These studies also demonstrated that MRD was 
detectable by the Signatera assay up to 16.5 months earlier than clinical relapse (on 
average, 8.7 months earlier) and that MRD clearance during treatment was a very strong 
predictor of patient response and long-term survival. Based on this performance, this test is 
well positioned to support escalation of therapy in the adjuvant setting.  
For description of methodological details regarding the Signatera assay for ctDNA 
analyses see reference 22 including Supplement. 


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1.3 
EO2040 a microbiome-derived therapeutic vaccine for treatment of minimal 
residual disease of colorectal cancer 
Summarized information about the EO2040 compound, and its targets are presented below; 
for more details see the current Investigator’s Brochure (IB) of EO2040 further describing 
the background for, and development of EO2040. 
Extensive pre-clinical studies have been conducted to characterize the microbiome-derived 
mimicry therapeutic vaccine concept (Figure 2), currently including two multi-peptide 
compounds in clinical trials (EO2401 in glioblastoma, adrenocortical carcinoma, and 
malignant pheochromocytoma/paraganglioma, and EO2463 in follicular and marginal zone 
lymphoma), and one compound in preparation for clinical trials (EO4010; targeted for 
multiple solid tumor types, including breast, colorectal and non-small cell lung cancer). 
EO2040, the compound to be used in the current trial is a derivative of the compound 
EO2401. EO2040 include the same components as EO2401, except the microbiome derived 
peptide EO2316, mimicking an epitope on the TAA interleukin 13 receptor alpha-2 (IL-
13Rα2). The reason to exclude peptide EO2316 from compound EO2040 is that IL-13Rα2 
currently does not have a rationale to be used when colorectal cancer is targeted, i.e. 
expression levels as known today are not high enough.  
Note, the parent compound of EO2040, i.e. EO2401, in combination with nivolumab, has 
been thoroughly investigated in two phase 1/2 trials in more than 100 patients with 
glioblastoma and adrenal tumors, showing the ability to generate strong systemic immune 
responses against the targeted TAAs, and being well tolerated; the safety profile being 
consistent with the profile of the combination partner nivolumab, with the only addition of 
local administration site reactions (most commonly grade 1, erythema, induration, and 
sometimes pain). 
The microbiome-derived mimicry concept builds on utilization of peptide-based 
vaccination, an immunotherapeutic approach for the treatment of cancer that aims to deliver 
immunogenic peptides corresponding to specific tumor associated antigens (TAAs) to 
patients. The goal is to target the patient’s antigen presenting cells (APCs), especially the 
dendritic cells (DCs), to induce efficient presentation of cancer epitopes to T lymphocytes 
that in turn leads to a sustained immune response against cancer cells expressing the targeted 
antigens at the tumor site. In the past, despite promising pre-clinical results in animal models, 
the cancer vaccination approach has not demonstrated unequivocal efficacy in patients. The 
lack of efficacy may be related to several factors including the status of the patient’s immune 
system, the efficacy and specificity of antigen delivery, the lack of ability of T lymphocytes 
to infiltrate the tumor microenvironment and the immunosuppressive environment, for 
instance due to the expression of inhibitory checkpoint molecules. The ability of tumor 
antigens to generate a strong immune response depends on several factors including the 
affinity for the MHC class I or MHC class II complexes, the capacity of the antigen to be 
recognized by the immune system as self or non-self, and pre-existence of T-cell clones that 
can be efficiently reactivated by a vaccine boost.  
EO2040, the microbiome-derived mimicry peptide mix for treatment of minimal residual 
disease of colorectal cancer, was developed to maximize the ability to generate a strong 
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 high affinity MHC class I binding-peptides (i.e. targeting efficacious presentation for 
CD8+ cytotoxic T cells),  
 high capacity to be recognized by the immune system by its non-self-nature, and  
 possibility for fast expansion of T cells by the pre-existence of T cell clones already 
recognizing the mimicry peptides which can be efficiently reactivated by a vaccine 
boost leading to a durable immune response.  
In addition, the "off-the-shelf" approach minimize treatment delays due to need of producing 
patient specific vaccines. 
The concomitant administration of EO2040 and an anti-PD1 blocking agent is utilized to 
optimize the possibility for tumor specific T cells to infiltrate and effectively function within 
the tumor environment (see further Section 1.3.5).  
Figure 2 : Microbiome-derived peptide therapeutic vaccine concept 
 
EO2040 Drug Product (DP; not adjuvanted) is a water/dimethyl sulfoxide (DMSO) peptide 
mixture solution of two synthetic 9-amino acid microbiome-derived peptides (EO2317 and 
EO2318), and the synthetic 15-amino acid helper peptide (Universal Cancer Peptide 2, 
UCP2). The peptide mix EO2040 (final concentration of each peptide is 300 µg/mL), will 
be emulsified with the adjuvant Montanide ISA 51 VG to reach a water in oil emulsion 
before subcutaneous (SC) administration. EO2040 will also be given in combination with 
the anti-PD1 monoclonal antibody nivolumab delivered according to global labels. 


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The EO2317 and EO2318 peptides were found in the human microbiota, are restricted to 
HLA-A2 expressed in 49% of the Caucasian population [24] and display high sequence 
homologies with two different TAAs: survivin also called baculoviral inhibitor of apoptosis 
repeat-containing protein 5 (BIRC5), and forkhead box M1 (FOXM1), respectively.  
These TAAs are highly expressed in colorectal cancer. The use of two different TAA targets 
were selected to overcome possible tumor heterogeneity and reduce tumor escape; both 
peptides demonstrated high MHC binding affinity, strong immune responses as well as cross 
reactivity against the human corresponding peptides in nonclinical models.  
1.3.1 
EO2317 and BIRC5/survivin  
EO2317 is a 9-amino acid microbial peptide with homology to a cognate T-cell epitope on 
BIRC5. In vitro binding assays showed high binding affinity of EO2317 for HLA-A2. In a 
vaccination study in HHD DR3 mice and HHD DR1 mice, EO2317 induced higher 
immunogenicity in the ELISpot IFN-γ assay compared with the human BIRC5 peptide and 
cross-reactivity with the corresponding BIRC5 human peptide was demonstrated. 
BIRC5, a well-known cancer driver gene, is a member of the inhibitor of apoptosis (IAP) 
gene family, which encodes negative regulatory proteins that function as endogenous 
inhibitors of caspases preventing apoptotic cell death [25]. BIRC5 also has a role as a mitosis 
regulator, physically associated with the mitotic apparatus thereby ensuring the proper 
completion of various stages of cell division probably via the regulation of microtubule 
dynamics and stability [26]. BIRC5 expression is high during fetal development and in most 
tumors, and absent to low in almost all adult tissues. 
BIRC5 Expression in normal human tissues (see Figure 3): 
While BIRC5 is strongly expressed in embryonic tissues where it plays a role in 
development, expression of BIRC5 in normal adult tissue is considered to be scarce. BIRC5 
expression is developmentally regulated and has been reported to be low expressed in most 
terminally differentiated normal human tissues [27]. More recent studies using more 
sensitive methods have however revealed that some adult tissues could express BIRC5 albeit 
at levels lower than cancer cells. In adult tissues, BIRC5, known to regulate adult stem cell 
physiology, is expressed, and regulated in normal tissues characterized by self-renewal and 
proliferation as in hematopoietic stem cells, keratinocytes stem cells or intestinal stem cells 
[28]. 
Analysis of gene expression databases shows moderate expression of BIRC5 in testis and 
thymus with sometimes also low expression in lymph nodes, colon, and esophagus [29, 30, 
31]. Massive peptide sequencing does not allow to detect specific BIRC5 peptides in normal 
tissues [32]. Other large-scale data available today on protein expression highlight the 
preferential expression of BIRC5 in the testis, with a low expression in the skin, oral mucosa, 
esophagus, and lymph nodes [31].  
 
 


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Figure 3 : Relative BIRC5 mRNA expression in normal tissues; (HPA RNA-seq normal 
tissues [29]; RPKM: Reads Per Kilobase Million) RNA-seq performed on 
tissue samples from 95 human individuals representing 27 different tissues 
 
BIRC5 Overexpression in colorectal tumor: 
As being able to control and regulate several pathways linked to tumorigenesis including 
protection from apoptosis and cell cycle promotion, BIRC5 is currently considered as a 
major tumor driver [33]. BIRC5 molecular functions are supported by a number of in vitro 
and in vivo preclinical studies that demonstrated that modulation of BIRC5 expression 
reduced tumor-growth, increased apoptosis and sensitized tumor cells to chemotherapeutic 
drugs [34]. Overexpression of BIRC5 in cancer overcome an apoptotic checkpoint and 
favour aberrant progression of transformed cells through mitosis.  
BIRC5 has been defined as a universal tumor antigen. BIRC5 gene expression profiles across 
tumor samples and paired normal tissues (from the TCGA and the GTEx projects), highlight 
the clear overexpression of BIRC5 in many tumor types, including glioblastoma, colon 
adenocarcinoma, lung, or breast cancer [35]. Studies presented in the literature studies also 
demonstrate overexpression of BIRC5 in almost all type of neoplasms, including lung, colon, 
breast, pancreas, stomach, liver, ovary and prostate cancer, melanoma, hematopoietic 
malignancies, and glioma [36, 37].  
In colon cancer, expression of BIRC5 is clearly documented. BIRC5 shows no or very low 
expression in normal colon tissues while BIRC5 is expressed in colon polyps and 
overexpressed in colonic adenocarcinoma [38]. Overexpression is observed at both the 
mRNA and protein levels with overexpression often seen in 80% to 100% of analyzed 
samples [38, 39, 40, 41]. Survivin expression in colon cancer is correlated with the degree 
of differentiation, depth of invasion, lymph node metastasis and Duke’s stage [42, 43], and 
is associated with poor prognosis (Figure 4) [43, 44]. Interestingly, BIRC5 overexpression 
is also described in colorectal cancer stem cells [108, 109], and even proposed as a biomarker 
to characterize circulating tumor cells and predict OS in mCRC patients [110, 111].  
 
 


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Figure 4 : Kaplan-Meier survival curves for PFS (A) and OS (B) in patients with stage 
IV colorectal cancer (N=71) [44] 
 
Overall, the large pattern of overexpression of BIRC5 in tumor, associated with its critical 
role in tumorigenesis makes BIRC5 an interesting chemotherapy 45], and immunotherapy 
target [47]. BIRC5 expression was shown to be significantly correlated with multiple 
immune cells infiltrates in a variety of tumor, and thus proposed as prognostic biomarker 
associated with tumor immune cell infiltration [37]. Furthermore, detection of BIRC5 
specific T-cells in peripheral blood from patients with melanoma, lymphoma [47], glioma 
[48], and colorectal cancer [49, 50, 51], further supports BIRC5 as a viable target for vaccine 
approaches. 


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Figure 5 : BIRC5 gene expression profile across all tumor samples and paired normal 
tissues; RNA sequencing expression data of 9,736 tumors and 8,587 normal 
samples from the TCGA and the GTEx projects [35] 
 
Figure 6 : BIRC5 gene expression profile across all colorectal samples and paired normal 
tissues; RNA sequencing expression data of BIRC5 in colon adenocarcinoma 
(COAD), rectum adenocarcinoma tumors (READ) and related normal 
samples from the TCGA and the GTEx projects [35] 
 


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In conclusion, BIRC5 has a very high expression in cancer cells as compared to the very low 
or absent expression in normal tissues (except the immune privileged testis and some 
lymphocytes), and thus BIRC5 is considered as an attractive anticancer target (see Figure 5 
and Figure 6).  
Based on overexpression in tumoral tissues and low/absent expression in normal tissues, 
BIRC5 was considered since a long time as an ideal target for vaccine or therapeutic 
approaches. Several strategies have been developed to target BIRC5 and have been tested in 
many clinical trials in many cancer types. This includes cell-based strategies such as BIRC5 
pulsed dendritic cells, and BIRC5 peptide vaccines. The safety information regarding the 
clinical trials targeting BIRC5 is summarised in the current IB of EO2040; in conclusion, 
there were no treatment limiting adverse events associated with the targeting of BIRC5. 
For clinical experience regarding the specific microbiome-derived peptide EO2317, see 
protocol Section 1.3.2. 
1.3.2 
EO2318 and FOXM1  
EO2318 is a 9-amino acid peptide identified from the human gut microbiome that targets a 
cognate T-cell epitope on FOXM1. In vitro binding assays showed high binding affinity of 
EO2318 for HLA-A2. In a vaccination study in HHD DR3 mice and HH DR1 mice, EO2318 
induced higher immunogenicity in the ELISpot IFN-γ assay compared with the human 
FOXM1 peptide and cross-reactivity with the corresponding FOXM1 human peptide was 
demonstrated. 
FOXM1, a member of the Fox transcription factors, regulates transcription of cell cycle 
genes essential for G1-S and G2-M progression, chromosome stability and segregation 
(including Nek2, KIF20A, CENP-A, AURKB, BIRC5, CENP-F, CDC25A, CDC25B, 
p27Kip1, cyclin B, and cyclin D1). FOXM1 is also involved in regulation of PI3K-AKT, 
TGF-β, Wnt/β-catenin, insulin, Sonic-Hedgehog, and Jagged-Notch signalling pathways. A 
current hypothesis is that abnormal activation of several kinases (such as MST1/2, RAF, 
RAS or MAPK2) leads to nuclear accumulation of FOXM1 in tumors. FOXM1 has been 
extensively studied and was shown to drive most of the hallmarks of malignancy. FOXM1 
exhibits a proliferation-specific expression pattern, and its expression is regulated by 
proliferation and anti-proliferation signals as well as by proto-oncoproteins and tumor 
suppressors [52]. FOXM1 is basically considered to be involved in all stages of oncogenesis 
[53, 54]. 
FOXM1 Expression in normal human tissues: 
FOXM1 is widely expressed in embryonic tissues and shows a low level of expression in 
normal adult tissues [55, 56]. Analysis of gene expression database shows remaining high 
expression of FOXM1 in the testis and low expression in hematopoietic cells, esophagus, 
colon, and skin [57, 58]. 


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The protein expression addressed by massive peptide sequencing does not allow to detect 
FOXM1 specific peptides in any tissues [32]. Other large-scale data of protein expression 
highlight the preferential expression of FOXM1 in the testis and hematopoietic cells.  
Figure 7 : Relative FOXM1 mRNA expression in normal tissues; (HPA RNA-seq normal 
tissues [59]; RPKM: Reads Per Kilobase Million) RNA-seq performed on 
tissue samples from 95 human individuals representing 27 different tissues 
 
FOXM1 Overexpression in colorectal tumor: 
While FOXM1 expression is turned off in terminally differentiated cells, it is upregulated in 
a multitude of human solid tumors including breast cancer, non-small cell lung carcinoma, 
hepatocellular carcinoma, pancreatic carcinoma, colon cancer, ovarian cancer, prostate 
cancer glioblastoma and others [60]. Gene expression profiles across tumor samples and 
paired normal tissues (from the TCGA and the GTEx projects), confirm the clear 
overexpression of FOXM1 in many different tumors including colorectal cancer [61]. This 
overexpression of FOXM1 was proposed to be a major predictor of adverse outcomes across 
39 human malignancies by computational analysis [62]. FOXM1 overexpression in 
colorectal cancer has been documented in many reports both at the mRNA and protein level, 
with overexpression observed in between 50 % to 85% of analyzed samples [63, 64, 65]. 
FOXM1 expression is directly linked to prognosis in colorectal carcinoma and FOXM1 
levels correlate with cancer progression, lymph node and liver metastasis, and high TNM 
stages [64, 66,67]. The important role of FOXM1 in metastasis development in CRC is 
thought to be linked to induction of the epithelial-mesenchymal transition [68, 69]. 
 
 


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Figure 8 : FOXM1 gene expression profile across all tumor samples and paired normal 
tissues; RNA sequencing expression data of 9,736 tumors and 8,587 normal 
samples from the TCGA and the GTEx projects [61] 
 
Figure 9 : FOXM1 gene expression profile across colorectal samples and paired normal 
tissues, 
association 
with 
overall 
survival 
in 
patients 
with 
colon 
adenocarcinoma; Kaplan–Meier analyses of overall survival according to 
FOXM1 expression levels in adenocarcinoma patients 70] 
 


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In conclusion, FOXM1 has a very high expression in cancer cells as compared to the very 
low or absent expression in normal tissues (except the immune privileged testis), and thus 
FOXM1 is considered as an attractive anticancer target.  
Despite the increased interest of FOXM1 as a target of antitumoral therapy [71, 72], to date 
no specific inhibitors are available for clinical studies, due to the poor druggability of this 
transcription factor.  
Interestingly, several FOXM1 peptides have been described for their ability to prime  
HLA-A2 or HLA-A24 restricted cytotoxic T lymphocytes in mice [73], suggesting that 
FOXM1 may be a suitable target for immunotherapy against cancers. Several phase I/II 
clinical trials were undertaken using FOXM1 peptides, for patients with HLA-A24 with 
gastric cancer, ovarian cancer, cervical cancer, or glioma. The safety information regarding 
the clinical trials targeting FOXM1 is summarised in the current IB of EO2040. 
For clinical experience regarding the specific microbiome-derived peptide EO2318, see 
protocol Section 1.3.2. 
1.3.3 
UCP2 – Helper peptide 
To generate an efficient immune response, the helper peptide UCP2 is included in EO2040. 
UCP2 was described by Godet [74]. It is a telomerase reverse transcriptase (TERT) derived 
CD4 epitope that binds to the most commonly found MHC class II alleles. This Th1 helper 
peptide should be able to expand T cells with the ability to sustain efficient dendritic cell 
activation and specific CTL activation by secreting interferon-gamma (IFN-γ), tumor 
necrosis factor-alpha (TNF-α) and interleukine-2 (IL-2) and enhancing expression of 
costimulatory signals on dendritic cells and T cells [75]. 
TERT expression is considered to be absent or extremely low in normal tissues (except in 
testis and some hematopoietic cells) while overexpressed in a large majority of tumors. The 
role of TERT in tumor progression is well documented. Cancer cells overcome senescence 
via telomere length maintenance mechanisms involving telomerase activation [76]. TERT 
overexpression achieved via multiple genetic and epigenetic mechanisms could be observed 
in 80–90% of malignant tumors [77, 78].  
The use of a helper antigen targeting TERT in colorectal cancer is further supported by the 
fact that telomerase activity and TERT expression were strongly enhanced in 80% to 100% 
of sample from adenocarcinoma tumors [79, 80, 81, 82], with the potential to be used as a 
biomarker and prognostic tool [83, 84].   
 


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Figure 10 : TERT gene expression profile across all tumor samples and paired normal 
tissues; RNA sequencing expression data of 9,736 tumors and 8,587 normal 
samples from the TCGA and the GTEx projects [85] 
 
The ability of UCP2 to elicit a specific CD4+ T cell responses has been demonstrated in in 
vivo models. Furthermore, spontaneous T cell responses against UCP2 were observed in 
various types of cancers [86], and this peptide is currently under clinical evaluation in phase 
1/2 studies in non-small cell lung cancer and in glioblastoma.  
Based on overexpression in tumoral tissues, low/absent expression in normal tissues and a 
major function in tumor development, TERT was considered since a long time as an ideal 
target for vaccine or therapeutic approaches.  
Several strategies have been developed to target TERT and have been tested in many clinical 
trials in many cancer types. This includes cell-based strategies such as TERT pulsed 
dendritic cells, TERT peptide vaccines, and adoptive transfer of T-cells. The safety 
information regarding the clinical trials targeting UCP2 is summarised in the current IB of 
EO2040. 
In the EO2040 peptide mix, UCP2 is mainly used as a target for helper T cells, to create T 
helper support for a cytotoxic response against the other targeted antigens and is not used as 
a target for cytotoxic cells. For clinical experience regarding the use of UCP2 in association 
with microbiome-derived peptides, see protocol Section 1.3.2. 


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1.3.4 
Montanide ISA 51 VG 
Montanide ISA 51 VG is an oily adjuvant composed of highly refined mannide oleate in a 
mineral oil solution. When mixed with an aqueous phase in a 50/50 ratio, it forms a water-
in-oil emulsion. This water-in-oil emulsion is used as vaccine adjuvant, to enhance the 
immune response. The effect of water in oil emulsions is thought to be linked with multiple 
processes. The main being the depot effect and the slow release of the antigen at the injection 
site, emulsions causing inflammation and the recruitment of antigen presenting cells, and 
they promote the uptake of antigens by antigen presenting cells. Clinical studies have been 
undertaken with Montanide ISA 51 VG since the 1990ties, mostly related to cancer and 
acquired immunodeficiency syndrome (AIDS). A recent example of the utilization of 
Montanide ISA 51 VG as an adjuvant for peptide therapeutic vaccination, also in 
combination with check point inhibition, is a phase 2 trial including patients with incurable 
human papilloma virus-16 related cancer [87].  
A review of safety of Montanide ISA 51 showed that generally observed adverse events 
(AEs) included injection site reactions, injection site pain, myalgia, headache, gastro-
intestinal disorders, fatigue, and fever, regardless of the administration route and patient 
characteristic [88]. Specific AEs, e.g. injection site reactions and rash, were more frequently 
reported from patients receiving Montanide ISA 51-adjuvanted vaccines than from patients 
receiving antigen, or Montanide ISA 51 only [88]. The reported AEs were mainly mild to 
moderate in intensity [88]. 
The EO2040 peptide mix, i.e. drug product (DP), will be emulsified with the adjuvant 
Montanide ISA 51 VG to reach a water in oil emulsion before subcutaneous (SC) 
administration. 
1.3.5 
Combination with anti-PD1 blockade 
The concomitant administration of EO2040 and an anti-PD1 blocking agent is utilized to 
optimize the possibility for tumor specific T cells to infiltrate and effectively function within 
the tumor environment.  
During the initial development of EO2401 for the treatment of solid tumors, nivolumab has 
been used in combination with EO2401 as the combination of a cancer vaccine and 
checkpoint inhibitors may function synergistically to induce more efficient anti-tumor 
immune responses [95, 96].  
The mechanism of action of anti-PD1 like nivolumab, blocking interaction between PD-1 
and its ligands, releasing PD-1 pathway-mediated inhibition of the immune response, 
including the anti-tumor immune response aims at facilitating access to the tumor by the T 
cells reactivated and expanded by the microbiome-derived therapeutic vaccine. The final 
objective being to induce a fast and extensive expansion and infiltration of tumor by the 
TAA specific T-cells. 
Except the strong general rationale combining anti-PD1 blockade with a vaccination 
approach [97, 98, 99], the rationale for the approach in the current study is also supported 
by the outcome of a mouse xenograft glioblastoma model utilizing a microbiome-derived 
peptide and anti-PD1 blockade (Figure 11).  


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Figure 11 : Mean tumor growing curves of U87MG glioblastoma engrafted mice 
following adoptive transfer of T cells from HHD DR3 mice vaccinated with 
EO2315 or human peptide, w/o combination with anti-PD1 antibody 
Note: EO2315 is a microbiome-derived peptide, and proof of concept progenitor part of EO2401. 
Untreated mice (1), Anti PD1 isotype control (2), Anti PD1 isotype control plus T-cells from EO2315 
immunized mice (3), Anti PD1 (4), Anti PD1 plus T-cells from human peptide immunized mice (5), Anti PD1 
plus T-cells from EO2315 immunized mice (6). 
A. results expressed as median; B. results expressed as mean plus SEM 
 
 
1.3.6 
Early clinical development of microbiome-derived peptides in solid tumors 
EO2040, the compound to be used in the current trial is a derivative of the compound 
EO2401. EO2040 include the same components as EO2401, except the microbiome derived 
peptide EO2316, mimicking an epitope on the TAA interleukin 13 receptor alpha-2 (IL-
13Rα2). The reason to exclude peptide EO2316 from compound EO2040 is that IL-13Rα2 
currently does not have a rationale to be used when colorectal cancer is targeted, i.e. 
expression levels as known today are not high enough.  
In the following, the parent compound of EO2040, i.e. EO2401, is described regarding early 
clinical development, especially with regard to safety, since it is assumed that the safety 
profile of EO2040 will be at pair with, or better than for EO2401. Note, per end-January 
2022, more than 100 patients with glioblastoma and adrenal tumors had started treatment 
with EO2401 in combination with nivolumab showing the ability to generate strong systemic 
immune responses against the targeted TAAs and being well tolerated; the safety profile 
being consistent with the profile of the combination partner nivolumab, with the only 
addition of local administration site reactions (most commonly grade 1, erythema, 
induration, and sometimes pain). 
The initial clinical development of EO2401 include two phase 1/2 trials, one in glioblastoma 
(EOGBM1-18; EudraCT# 2018-002279-16, IND# 19229) and one in adrenal tumors 
(EOADR1-19; EudraCT# 2019-003396-19, IND# 19229). 
The early clinical development of EO2401 aimed at showing that therapeutic cancer 
vaccination with EO2401 in combination with anti-Programmed cell Death protein 1(PD1) 
Tumor size (mm3)
p=0.04
p=0.0004
p<0.0001
A
25
30
35
40
45
50
0
250
500
750
1000
1250
1500
1750
2000
2250
Time after implantation (days)
1-Untreated
2-Anti-PD1 isotype control
3-Anti-PD1 isotype control plus T cells from EO2315 immunized mice
4-Anti-PD-1
5-Anti-PD1 plus T cells from Human peptide immunized mice
6-Anti-PD1 plus T cells from EO2315 immunized mice
B


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blockade is safe and tolerable, and can achieve expansion of T cells not only recognizing the 
microbiome derived peptides used for immunization but also recognizing the targeted 
nominal TAAs expressed by human tumor cells in patients with malignancies (i.e. 
immunological proof of concept). In addition, preliminary efficacy aspects of EO2401 as 
well as multiple translational exploratory endpoints are included in the early development 
trials.  
The first-in-human (FIH) trial of EO2401, EOGBM1-18, is a multicenter (10 sites in France, 
Germany, Spain, and USA), phase 1/2 trial, in patients with unequivocal evidence of 
progressive or first recurrent glioblastoma. The study design builds on an initial cohort for 
assessment of mainly safety and tolerability of monotherapy EO2401, and EO2401 in 
combination with nivolumab (a cohort with a 3-by-3 design), and expansion cohorts for 
assessment of further safety and tolerability, immunogenicity, and preliminary efficacy 
during treatment with EO2401 in combination with nivolumab (without any EO2401 
monotherapy component), and EO2401 in combination with both nivolumab and 
bevacizumab concomitantly (the latter treatment option only available for sites in the USA 
due to local health authority authorization of bevacizumab for recurrent glioblastoma). The 
study was initially planned to enroll a maximum of 52 patients but has since been expanded 
to approximately 80 patients, and the first patient treated in trial EOGBM1-18 received 
EO2401 monotherapy on July 28, 2020. See Section 1.3.6.1 for preliminary outcome-
information related to trial EOGBM1-18.  
The second early clinical development trial including EO2401, EOADR1-19, is a 
multicenter (10 sites in Denmark, France, Germany, Italy, Netherlands, Spain, Sweden, and 
USA), phase 1/2 trial, in patients with adrenocortical carcinoma or malignant 
pheochromocytoma/paraganglioma. The study design builds on an initial cohort for 
assessment of safety and tolerability of EO2401 in combination with nivolumab (a cohort 
with a 3-by-3 design), and expansion cohorts for further assessments of safety and 
tolerability, immunogenicity, and preliminary efficacy in specific subgroups of patients with 
adrenal tumors (previously untreated, or previously treated, with each adrenal tumor entity). 
The study was initially planned to enroll 60 patients in the expansion phase cohorts after the 
initial safety cohort was finalized but is now to be expanded beyond this with a randomized 
component including 65 patients with previously treated adrenocortical carcinoma. The first 
patient treated in trial EOADR1-19 received EO2401 in combination with nivolumab on 
August 11, 2020.  
The trials EOGBM1-18 and EOADR1-19 are overseen by independent data monitoring 
committees (IDMCs) which are cross-sharing information.  
For completeness, it should be mentioned that the microbiome-derived mimicry therapeutic 
vaccine concept is also applied with a second compound, EO2463, targeting B cell markers, 
in a clinical trial in follicular and marginal zone lymphoma (EONHL1-20; EudraCT 2020-
003999-40). The first patient treated in trial EONHL1-20 received EO2463 monotherapy on 
July 5, 2021. Even if clear inter-study differences regarding populations/compounds, the 
IDMC for trial EONHL1-20 is also participating in the cross-sharing of information with the 
IDMCs for trials EOGBM1-18 and EOADR1-19 to enhance the possibility to detect possible 
concept related safety signals. No such signal has been found currently. 
1.3.6.1 
EO2401 clinical safety experience  
Two safety assessments have been performed to define the early development safety profile 
of EO2401 in combination with nivolumab +/- bevacizumab: 


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 A protocol pre-planned interim assessment of trial EOGBM1-18, with a clinical cut-
off 2021-OCT-29.  
 A planned safety assessment of trial EOADR1-19, with a database cut-off 2021-
NOV-30.  
Summaries and conclusions of the full assessments are given below. 
Of note, based on IDMC assessments, the safety lead-in parts of both trials (EOGBM1-18 
and EOADR1-19) have run with a minimum number of patients (each 3 patients) due to lack 
of safety issues (no safety concern events detected), and all expansion cohorts in both trials 
have, after further IDMC assessments, been opened as planned for recruitment.  
The dose and schedule established for EO2401 based on studies EOGBM-18 and EOADR1-
19 and IDMC recommendations, is 300 µg/mL of each of the peptides in the mix per dose 
administration (i.e. 1.2 mg/mL protein per administration with the included 4 peptides), and 
administration by 4 priming injections SC at 2-weekly intervals, followed by boosting 
injections starting at 4 weeks after the fourth priming injection; continued boosting injections 
given on a 4-weekly interval. 
The EO2401 dose and schedule per above have been used in combination with nivolumab, 
and in combination with nivolumab/bevacizumab.  
The nivolumab dose and schedule applied in trial EOGBM1-18 followed the early 
development principles for nivolumab, i.e. a dose of 3 mg/kg administered every 2 weeks in 
combination with EO2401. For trial EOADR1-19 the more convenient, and also labelled, 
dose/schedule instruction was: 
 Nivolumab is to be administered as an IV infusion starting from the first priming 
injection of EO2401, and then following the schedule in priming and boosting, of 
EO2401. The nivolumab infusion is to start 3 hours after the EO2401 administration. 
The dose of nivolumab should be 240 mg every 2 weeks for the first 3 
administrations, and from the fourth administration, and onwards, a nivolumab dose 
of 480 mg every 4 weeks is to be applied. 
Note, all patients have received EO2401 by SC administration together with the adjuvant 
Montanide ISA 51 VG.  
Summary of safety of EO2401 in combination with nivolumab +/- bevacizumab in 
patients with recurrent glioblastoma 
Trial EOGBM1-18 includes Cohort 1 (safety lead-in; patients with first recurrent measurable 
disease; treatment EO2401 for 4 weeks, then switch to EO2401/nivolumab if treatment 
tolerated), Cohort 2a (patients with first recurrent measurable disease; treatment 
EO2401/nivolumab), Cohort 2b (patients with non-measurable disease; adjuvant treatment 
after resection for first recurrent disease; treatment EO2401/nivolumab), and Cohort 3 
(patients 
with 
first 
recurrent 
measurable 
disease; 
treatment 
EO2401/nivolumab/bevacizumab).  
At the clinical cut-off a total of 40 patients had started study treatment including 3 patients 
in Cohort 1, 23 patients in Cohort 2a, 3 patients in Cohort 2b, and 11 patients in Cohort 3. 
The median age of patients treated in the study (n=40) was 60.0 years (range 24-78 years), 
including 19 (47.5%) female patients and 21 (52.5%) male patients. 


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Extent of exposure 
Per the clinical cut-off 2021-OCT-29, 12 of the patients had ongoing study treatment, and 
28 had terminated study treatment due to the following reasons: 
 24 patients due to progressive disease. 
 1 patient due to "lost to follow-up". 
 2 patients due to "other" reasons ("decision for discontinuation by the patient", and 
"clinical progression", respectively). 
 1 patient due to adverse event (transaminitis Grade 4 judged as possibly related to 
nivolumab). 
The median overall duration of exposure was 2.33 months, however, showing distinct 
differences between cohorts (Cohort 1 > Cohort 2b > Cohort 3 > Cohort 2a), especially 
considering the fact that recruitment start to Cohort 3 was later than to other cohorts, and 
that most patients in that cohort still are on treatment. The longest individual treatment 
duration is noted in Cohort 1, i.e. a treatment duration of 14.3 months, and still ongoing at 
the clinical cut-off. 
Adverse events 
The most common TEAEs (cut at 10% among 40 patients), irrespective of relationship are: 
headache (38%), fatigue (30%), injection site reaction (15%), and nausea (15%) (Table 1). 
The most common treatment related TEAEs (cut at 5% among 40 patients) are: fatigue 
(23%), injection site reaction (13%), and nausea (10%) (Table 2). 
Table 1 : The most common adverse events (cut at 10%), irrespective of relationship 
noted in trial EOGBM1-18  
 
 
 
 


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Table 2 : The most common treatment-related adverse events (cut at 5%), noted in trial 
EOGBM1-18 
 
The majority, 32 of 40 (80%), of patients have experienced no, or only mild (Grade 1) to 
moderate (Grade 2), adverse events. Grade 3/4 events, irrespective of relationship, were seen 
in 20% of patients, and related Grade 3/4 events in 7.5% of patients. No Grade 5 event was 
observed. 
Immune-mediated events 
 Alanine and aspartate aminotransferase increases:  
o Two patients have had what has been interpreted as immune-mediated 
hepatitis leading to interruption of EO2401/nivolumab (resolved without 
sequelae) and discontinuation of EO2401/nivolumab/bevacizumab (ongoing 
but slowly normalizing lab values, follow-up ongoing at the clinical cut-off); 
both patients have received steroids during the events. 
o In addition to above, and not yet documented in the eCRF by the clinical cut-
off is the discontinuation of nivolumab at a single value Grade 3 ALT in one 
patient (in accordance with protocol EOGBM1-18 version 3). 
 Blood thyroid stimulating hormone changes:  
o One patient has experienced an immune-mediated, initial hyperthyroidism 
(Grade 1), followed by hypothyroidism (Grade 2), requiring substitution 
therapy with L-thyroxin. 
o Further three patients have experienced treatment related thyroid stimulating 
hormone changes without the need for therapy; two patients with increased 
laboratory values, and one patient with a decreased laboratory value. 
   Adrenal insufficiency: 
o Two patients have reported adrenal insufficiencies Grade 1 (diagnostic 
observations only; intervention not indicated); one patient with no change of 


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study treatment, no therapy for the event, and resolved without sequelae, start 
day 15, end day 56 (note, patient also had a s-cortisol below Lower Limit of 
Normal (LLN) at baseline), and one patient with no change of study 
treatment, no therapy for the event, not resolved, start day 16, study treatment 
ongoing at the clinical cut-off (baseline s-cortisol was within normal limits). 
Considering the description of the cases, and also considering the situation 
around treatment of recurrent glioblastoma (e.g. use of steroid treatments), 
the above cases are for the time being considered as possibly immune-
mediated pending further assessment. 
Clinical laboratory assessments 
The interim assessment clearly indicates that there is no significant level of hematological 
toxicity linked to treatment with EO2401/nivolumab or EO2401/nivolumab/bevacizumab.  
Likewise, the assessment clearly indicates that there is no significant level of non-described 
toxicity regarding biochemistry parameters linked to treatment with EO2401/nivolumab or 
EO2401/nivolumab/bevacizumab, beside the already known immune-mediated hepatitis 
giving rise to transaminitis, linked to nivolumab treatment.   
Administration site reactions 
The most distinct event type linked to EO2401 (and the SC application together with the 
adjuvant Montanide ISA 51 VG) is administration site reactions. At the time of the clinical 
cut-off adverse administration site reactions have been reported as TEAEs in 19 (47.5%) of 
40 treated patients. These patients experienced in total 38 events assessed as being 
administration site reactions (median 1 event/patient, average 2 events/patient, range 1 to 7 
events/patient). Only 2 (5.3%) of the events led to concomitant drug treatment, in both cases 
with paracetamol. 
The median time to first onset of an administration site event in a patient (n=19 patients) was 
16 days (range 1 to 124 days), and the median time to onset of any event (n=38 events) was 
35.5 days (range 1 to 446 days).  
At the clinical cut-off 28 of the 38 events had been resolved without sequelae, and 10 events 
were still ongoing. The median duration of the 28 resolved events was 19.5 days, and the 
mean duration was 43.25 days (range 1 to 211 days). The median duration of all 38 events 
utilizing a Kaplan-Meier analysis was 42 days (range 1 to 431 days) censoring duration at 
the clinical cut-off (Figure 12). Of note, at the time of the interim assessment there was no 
correlation between administration site reactions and clinical outcome, but it can be noted 
that the longest duration event is present in the patient with the longest, also ongoing, study 
treatment (also confirmed 82%, i.e. partial, recurrent glioblastoma regression from baseline). 
Note, due to the reporting structure the duration of a local administration site event might 
include durations at different administration sites. The location of EO2401 injections is 
rotating (so an injection site will be used every fourth time, i.e. during the booster period for 
EO2401, there will be 4 months between the utilization of one individual injection site), with 
the injection sites being right and left upper extremity and right and left lower 
extremity/inguinal area. The rotation system has made it possible, also in patients with the 
most extensive administration site reactions, to continue EO2401 administrations without 
any interruptions due to this cause. 


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Figure 12 : EOGBM1-18: Duration of local administration site reactions per clinical 
cut-off 2021-OCT-29 based on 38 events reported in 19 patients (of a total of 40 
patients) 
 
Of the 38 events, 32 (84.2%) were of Grade 1, and 6 (15.8%) of Grade 2. Of the 19 patients, 
15 (37.5% of 40 patients) had a maximum of Grade 1, and 4 (10% of 40 patients) had a 
maximum of Grade 2. 
Three of the Grade 2 events are resolved without sequelae (starting study day/duration: start 
44/ duration 3, start 151/duration 25, and start 373/ duration 49 days, respectively), and 3 
events are still ongoing (starting study days 211, 220, and 446, respectively). 
The type of administration site reactions noted have been (note, among 38 events there are 
6 events with two types of reactions described, thus the denominator below is 41 reactions): 
 Erythema (29.3%) 
 Reaction, without further specification (22.0%)  
 Induration (19.5%) 
 Pain (17.1%) 
 Hematoma (7.3%) 
 Intermittent secretion at injection site (2.4%) 
 Pruritus (2.4%) 
In conclusion, the combination of EO2401 (administered SC with the adjuvant Montanide 
ISA 51 VG) and nivolumab +/- bevacizumab used for treatment of patients with recurrent 
glioblastoma, is well tolerated with a safety profile consistent with the safety profile of 
nivolumab monotherapy, and when applicable the safety profile of bevacizumab 
monotherapy, except the addition of local administration site reactions. Any local 


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administration site event (main types are erythema, induration, or pain) was seen in 48% 
of patients with a median time to onset of 16 days (range 1 to 124 days), and a median 
duration in currently resolved events (74% of all events) of 20 days (range 1 to 211 days). 
Maximum grade of the events was Grade 1 in 15 (38%) patients, and Grade 2 in 4 (10%) 
patients; no Grade ≥ 3 was encountered in trial EOGBM1-18. 
Of note, administration site reactions seen in trial EOGBM1-18 are below, or well within 
the expected frequency for any antigen administered together with Montanide ISA 51 VG 
as an adjuvant [88]. van Doorn et al. indicate injection site inflammation, edema and redness, 
damage, or induration in 40% to 100% of patients in randomized trials; assumed around 70% 
by SC route in non-randomized trials [88]. 
Summary of safety of EO2401 in combination with nivolumab in patients with adrenal 
tumors 
Trial EOADR1-19 includes Cohort 1 (safety lead-in; patients with adrenal tumor and 
previously treated disease; treatment EO2401/nivolumab), Cohorts 2A/B (patients with 
adrenocortical 
carcinoma), 
and 
Cohorts 
3A/3B 
(patients 
with 
malignant 
pheochromocytoma/paraganglioma); A, denotes patients with previously treated disease; 
and B, patients with no previous treatment for advanced/metastatic disease. 
For trial EOADR1-19 the current safety assessment includes a total of 43 patients who had 
started study treatment, including 33 patients with adrenocortical carcinoma (ACC; Cohorts 
2A/2B) and 10 patients with malignant pheochromocytoma/paraganglioma (MPP; Cohorts 
3A/3B).  
The median age of patients treated in the study was 48 years (range 20-85 years), including 
29 (67%) female patients and 14 (33%) male patients. The ECOG score was 0 in 54%, 1 in 
42%, and 2 in 5% of patients. Thirty-five percentage (35%) patients had metastatic 
involvement of one organ, 35% of two organs, 23% of three organs, and 7% of more than 
three organs. Liver metastases were present in 62% of patients at baseline, also reflected by 
19% of patients having baseline increases of aspartate aminotransferase and gamma-
glutamyltransferase, and 12% having increases of alanine aminotransferases. 
Eleven of the patients had ongoing study treatment (up until the visit at week 39 per DB 
status at the cut-off), and 32 had terminated treatment, at the time of the DB cut-off.  
The most common adverse events, irrespective of relationship, were pyrexia (30%), diarrhea 
(26%), fatigue (21%), and aspartate aminotransferase increased (21%) (Table 3), and the 
most common related adverse events were fatigue (19%), injection site pain (12%), pyrexia 
(12%), and diarrhea (12%) (Table 4). 
Grade 3/4 events, irrespective of relationship, were seen in 47% of patients, and related 
Grade 3/4 events in 12% of patients (9 events in 5 patients; ALT increased, injection site 
pain, oral mucositis x 2/Injection site reaction x 2/fever, auto-immune hepatitis, 
lymphopenia). Of note, further and final assessment by investigators, in relation to data 
cleaning, of the related events might lead to adjustment of the events in some of the patients.  
Two patients, one each with ACC and MPP, respectively terminated study treatment due to 
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Table 3 : The most common (cut at 10% among 43 patients) TEAEs by MedDRA 
preferred term irrespective of relationship in trial EOADR1-19 (Cohorts 2 and 3, 
combined cohorts) [extracted from Table 14.3.3.2.3] 
 
Table 4 : The most common (cut at 5% among 43 patients) TEAEs related to study 
treatment by MedDRA preferred term in trial EOADR1-19 (Cohorts 2 and 3, 
combined cohorts) [extracted from Table 14.3.3.3.3] 
 
TEAEs assessed to be immune-mediated events include the following cases:  
 Patient NL01-06: developed immune-mediated hepatitis (autoimmune hepatitis; related 
Grade 3) day 43 leading to study drug interruption and necessitating high-dose steroid 
treatment; study treatment was withdrawn approximately 2 months later due to 
progressive disease and the patient is alive more than 5 further months later (remaining 
event reported day 92 is "Decreased liver function" Grade 1, not resolved). 
 In addition, there are also three patients where hyper/hypothyroidism have been 
recorded, all events considered related to nivolumab only: 
o NL01-10; hyperthyroidism Grade 1, day 70, resolved day 127 without any 
treatment. 
o SE01-02; hyperthyroidism Grade 1, day 42, ongoing untreated. 


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o DE01-02; hyperthyroidism Grade 1 day 71 to 127, thereafter switch to 
hypothyroidism Grade 2, ongoing with L-thyroxine substitution. 
There has not been any death during the study which has been judged as related to study 
treatment. Four AEs are indicated to be leading to death; these were non-related events of 
COVID-19 infection, death, euthanasia, and cardiac insufficiency. The AE of "Death" in 
patient IT01-18 is under clarification since the primary reason for death is recorded in the 
eCRF as progression of ACC. 
The most distinct event type linked to EO2401 (and the SC application together with the 
adjuvant Montanide ISA 51 VG) is administration site reactions. At the time of the clinical 
cut-off adverse administration site reactions have been reported (or is assumed) as TEAEs 
in 15 (35%) of 43 treated patients.  
The median time to onset of an administration site event (n=31) was 29 days, and the mean 
time to onset was 43 days (range 2-195 days). Duration is not possible to fully assess 
currently since 20 of the 31 events still does not have a final evaluation of the treating 
physicians. However, for the 11 events with currently an event stop date there is a 
distribution indicating that most of the events might have a duration of less than 3 weeks 
(64%), an intermediate group might have a duration of approx. 5-6 weeks (27%), and a 
smaller proportion might have a longer duration (9%). Note, these estimates of event 
duration based on incomplete data should be interpreted with caution.    
The preferred terms of the events (total events n=31) are as follows: 
o Injection site reaction (29%)  
o Injection site pain (23%)  
o Erythema (13%) 
o Injection site induration (10%) 
o Injection site erythema (10%) 
o Skin reaction (10%) 
o Induration (6%) 
The NCI CTC grade per patient (n=15) reported currently are as follows: 
o Grade not reported (7%)  
o Grade 1 (67%)  
o Grade 2 (13%)  
o Grade 3 (13%)  
o Grade 4 (0%)  
Of note, the location of EO2401 injections is rotating (so an injection site will be used every 
fourth time - meaning when the events seem to be more frequently occurring, i.e. during the 
booster period for EO2401, there will be 4 months between the utilization of one individual 
injection site), with the injection sites being right and left upper extremity and right and left 
lower extremity/inguinal area. The rotation system has made it possible, also in the patient 
with the most extensive administration site reactions, to continue EO2401 administrations 
without any interruptions due to this cause.  
In conclusion, the combination of EO2401 (administered SC with the adjuvant Montanide 
ISA 51 VG) and nivolumab used for treatment of patients with adrenal tumors, including 
patients both previously treated, and previously systemically untreated for 
advanced/metastatic disease, is well tolerated with a safety profile consistent with the 
safety profile of nivolumab monotherapy, except the addition of local administration site 


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reactions (any local symptom of erythema, induration, pain, seen in 35% of patients with 
a median time to onset of 29 days; the majority (67%) of patients with a reaction had mild 
(Grade 1) events, but 2 patients experienced Grade 3 events). 
Of note, administration site reactions seen in trial EOADR1-19 are below, or well within the 
expected frequency for any antigen administered together with Montanide ISA 51 VG as an 
adjuvant [88].  
Overall conclusion regarding safety of EO2401  
Overall, taking into account the specifics of the differences in patient populations, the early 
development safety profiles for EO2401 in trials EOGBM1-18 and EOADR1-19, seems 
similar and showing that the compound is well tolerated with a safety profile consistent with 
the safety profile of nivolumab monotherapy (plus bevacizumab monotherapy when 
applicable), except the addition of local administration site reactions. 
It is currently assumed that the differences seen in some parameters describing local 
administration site reactions between trial EOGBM1-18 and EOADR1-19 are related to the 
sometimes-short follow-up and still ongoing treatments.  
1.3.6.2 EO2401 clinical efficacy and immunogenicity  
The key clinical efficacy parameter for the recurrent setting of glioblastoma (trial EOGBM1-
18), i.e. survival, had at the time of the interim analysis a median follow-up of 7.9 months 
(range 2.3-14.8 months) for patients treated with EO2401 in combination with nivolumab 
(Cohorts 1+2a+2b), and 3.2 months (range 2.2-6.0 months) for patients treated with EO2401 
in combination with nivolumab/bevacizumab (Cohort 3). Thus, from a main clinical efficacy 
perspective, i.e. survival, the interim analysis was immature, which is supported by the fact 
that the median survival had not been reached in any of the Cohorts, or overall, based on the 
then current 6 events and 34 censored patients. 
A survival update for patients in Cohorts 1/2a/2b (n=29), i.e. patients treated with EO2401 
in combination with nivolumab, was performed by a database cut-off 2021-DEC-01, at 
which time the median follow-up was 9.3 months (range 2.8 - 15.6 months). The updated 
analysis includes 10 events and 19 censored patients. The median survival has still not been 
reached for the patients treated in Cohorts 1/2a/2b, and the estimated survivals at 6 months 
(85.0%), 9 months (66.1%), and 12 months (50.1%; note, early assessment) seems to 
compare well with the expected survival of for instance lomustine which is a standard of 
care in the setting (lomustine estimated survival at 9 months 47.5%, and 12 months 34.1%, 
respectively; [89]).   
Overall, the disease control rate among 40 patients was 42.5%, including long duration 
partial responses (longest with treatment duration beyond 19 months), and one pathologic 
complete remission; a patient in Cohort 2a taken off study treatment at week 7 due to 
increased neurological symptoms and MRI progression of target lesion of 63%, where the 
patient one month later had surgery showing a complete pathological remission (only 
necrosis, extensive immune cell infiltration, and edema in the resected material = 
pseudoprogression seen on MRI).  
Early assessments of response and survival are interesting, however of key importance is 
that despite in many cases short study treatment durations, immune monitoring demonstrated 
the ability of EO2401 to induce a strong Tc1-skewed CD8+ T cell response with strong 


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cross-reactivity against the human targeted Tumor Associated Antigens (TAAs) (Figure 13 
and Figure 14). A positive response against the 3 bacterial peptides included in EO2401 was 
demonstrated for all patients investigated, and cross-reactivity against human targeted TAAs 
was demonstrated in 20 of 21 investigated patients using the IFN-g ELISpot assay. 
Importantly, for some patients, positive response was already detected two weeks after the 
first vaccination. 
Figure 13 : EOGBM1-18: EO2401 induces strong immune response in all patients. 
Tetramer staining after in vitro stimulation, Interpole hyperbola curve from all data 
points, 95% confidence bands. 
 
Figure 14 : EOGBM1-18: Cross-reactivity in patients demonstrated for all 3 TAAs 
(examples). 
 
In trial EOADR1-19, patients with malignant pheochromocytoma/paraganglioma due to the 
nature of their disease (neuroendocrine tumor) have been followed too short time to make 
an analysis relevant. Among patients with adrenocortical carcinoma follow-up is also too 
short to assess survival but considering progression-free survival and objective (RECIST 
1.1) response it has been possible to define a group of patients with a better-than-expected 
outcome of study treatment (disease control rate of 64%, and 6-months progression-free 
survival rate of 42%); adjusted inclusion/exclusion criteria are now to be further assessed in 
a randomized extension of the trial. Of note, usual checkpoint inhibitor "potential response 
influencing factors" does not seem to impact efficacy of the EO2401/nivolumab combination 
in patients with adrenocortical carcinoma; rather the best responding patients have low tumor 
mutational burden (TMB), MSI-low/MSS status, and low PD-L1 expression in their tumors.      
Immunogenicity, as far as tested in trial EOADR1-19, show strong CD8 T cell ELISPOT 
responses against the vaccine peptides (9/9 patients) and cross-reactivity against targeted 
TAAs (in 8/8 evaluable patients). Tetramer staining of specific CD8 cells for all 3 peptides 


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was detected in 7/8 tested patients; and when investigated, positive staining against BIRC5 
was detected as early as 4 weeks after the first vaccination. 
1.3.7 
Benefit/risk assessment and description of and justification for the route 
of administration, dosage, dosage regimen, and treatment period 
The trial is a multi-center, open-label, non-comparative, two sequential cohort, phase 2 trial, 
to investigate as the first cohort efficacy of the microbiome-derived therapeutic vaccine 
EO2040 in combination with nivolumab in patients with stage II-IV colorectal cancer with 
ctDNA defined MRD after completion of curative therapy. Assuming a positive outcome of 
the first cohort, a second cohort assessing efficacy of EO2040 monotherapy, with the option 
of addition of nivolumab after 3 months in case of no ctDNA clearance, is planned.  
The rationale for the proposed study is outlined in Section 2.1, indicating the current lack of 
therapy options for patients with ctDNA defined MRD after completion of all available 
standard of care curative treatments, and thus the need for new therapeutic approaches.  
Application of rationally developed immunotherapies might be one avenue to explore the 
possibility to enhance the survival chance for patients with ctDNA defined MRD 
recurrence/progression of CRC. An innovative option which has been tested in the clinical 
phase 1/2 setting and shown to have a predictable safety profile and indications for the 
possibility of efficacy in solid tumors, is the use of microbiome-derived peptide vaccination 
targeting tumor associated antigens (TAAs) highly expressed, and linked to clinical 
outcome, in CRC, in combination with anti-PD1 blockade in the form of nivolumab.  
EO2040, the compound to be used in the current trial is a derivative of the compound 
EO2401. EO2040 include the same components as EO2401, except the microbiome derived 
peptide EO2316, mimicking an epitope on the TAA interleukin 13 receptor alpha-2 (IL-
13Rα2). The reason to exclude peptide EO2316 from compound EO2040 is that IL-13Rα2 
currently does not have a rationale to be used when colorectal cancer is targeted, i.e. 
expression levels as known today are not high enough.  
The components of EO2040 are two microbial-derived peptides (EO2317 and EO2318) 
mimicking cytotoxic T cell (CD8+ T cell) HLA-A2 restricted epitopes from two different 
TAAs, BIRC5/survivin and FOXM1, respectively, and the helper peptide (CD4+ T cell 
epitope) universal cancer peptide 2 (UCP2), derived from hTERT.    
For details regarding the non-clinical safety evaluations of the microbiome derived 
therapeutic vaccine concept, including EO2040, see the current IB of EO2040, in summary: 
 Neither off-target (non-specific) nor other adverse effects that may occur following 
binding of the peptides to HLA molecules have been observed in HLA-A2 transgenic 
mice. 
 It has been demonstrated in wild type mice that activation of T cells by vaccination 
with mouse microbial-derived peptides does not cause toxic immune reactions 
against microbial gut contents; the obtained results suggest good tolerability and no 
obvious signs of toxicity.  
 Assessment of the safety of the concomitant administration of microbial-derived 
peptides with anti-PD1 showed that the combination of mouse bacterial peptides with 
anti-PD1 revealed no sign of toxicity, confirming that induction of a systemic 
immune response against a bacterial antigen does not affect gut homeostasis. 
The safety, and benefit/risk, profile of nivolumab is well known via the multiple labels for 
different malignant diseases already approved globally, also including a subgroup of patients 


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with CRC (mismatch repair deficient (dMMR) or microsatellite instability-high (MSI-H) 
metastatic disease after prior treatment) [90, 91]. 
The parent compound of EO2040, i.e. EO2401, in combination with nivolumab, has been 
thoroughly investigated in two phase 1/2 trials in more than 100 patients (by end-January 
2022) with glioblastoma and adrenal tumors, showing the ability to generate strong systemic 
immune responses against the targeted TAAs, and being well tolerated; the safety profile 
being consistent with the profile of the combination partner nivolumab, with the only 
addition of local administration site reactions (most commonly grade 1, erythema, 
induration, and sometimes pain). The clinical experience to date for EO2401 (administered 
with the oil-in-water adjuvant Montanide) in combination with nivolumab, and 
nivolumab/bevacizumab is described in Section 1.3.2. 
The overall assessment of the Sponsor is that the safety profile expected for the current trial, 
as already indicated by the experience in phase 1/2, of the combination of EO2401 with 
nivolumab, will be like the well described safety profile of nivolumab given without 
EO2040, with the addition of local administration site reactions (erythema, induration, and 
sometimes pain) in around half of the patients provided longer treatment durations are 
achieved. The median duration of local administration site reactions is expected to be as in 
trial EOGBM1-18, i.e. around 6 weeks, assumed without the need to adjust study treatment 
delivery due to the applied rotating administration sites per below.  
The location of EO2040 injections is rotating, i.e. an injection site will be used every fourth 
time, meaning that during the current trial with a pre-determined treatment duration of 
maximum 6 months, each administration site will only be used twice); the injection sites are 
right and left upper extremity, and right and left lower extremity/inguinal area, with the more 
specific locations being: 
 for upper extremity = area of armpit, or deltoid muscle of arm, depending on the 
constitution of the patient, and  
 for lower extremity = area of groin, or anterolateral thigh muscle, depending on the 
constitution of the patient. 
In addition to above, and as for all administrations of immunogens, it cannot be excluded 
that immediate type allergic reactions might occur, even if immediate type allergic reactions 
at injections of EO2401 has not yet been seen in any patient at any administration of EO2401 
during the early clinical development; special precautions and guidance are outlined for this 
type of potential reactions (Section 12.4). 
The selection of antigens targeted by EO2040 and their expression in normal tissues makes 
it unlikely that other normal cells, would be targeted by EO2040 expanded T cells; for further 
details see Sections 1.3.1 1.3.2, and 1.3.3, and the current version of the IB for EO2040. 
However, provided EO2040 induces an efficacious expansion of T cells specific for the 
targeted TAAs with ability to kill tumor cells expressing one, or both, of these TAAs, it 
cannot be ruled out that such antigen specific T cells might also recognize these antigens if 
expressed, and presented by MHC, at a high enough level (i.e. expressed as in tissue from 
CRC) on non-cancerous cells. Thus, if killing of tumor cells by T cells expanded by EO2040 
immunization could occur, normal cells (with the same high level of expression as CRC; i.e. 
unlikely situation - see above) might also be killed by such T cells leading to symptoms like 
for the immune-related adverse reactions which might be seen during treatment with anti-
PD1 (or similar compounds) alone (e.g. see the European SmPC or the US PI for nivolumab 
[90, 91]. It can be assumed that the immune-related adverse reactions seen during treatment 
with e.g. nivolumab are driven by self-reactive T cells, even if in the case of nivolumab the 


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specific targets for these T cells are not known due to the non-specific mode of action of 
nivolumab in relation to antigen-specificity. Thus, the mechanism for possible normal tissue 
toxicity by EO2040 immunization is assumed to be the same as for nivolumab induced 
immune-related toxicity and by that safety measures have been instituted which directly 
follows the measures already well established for nivolumab and similar compounds (see 
Section 6.4.1). Of note, the early clinical development of EO2401 does not indicate that there 
is an added frequency of immune-mediated adverse reactions by the combination of EO2401 
and nivolumab, as opposed to administration of nivolumab monotherapy; see Section 1.3.2 
[90, 91].  
Generally, anti-tumor therapeutic vaccination approaches tested to date have indicated the 
need for booster doses to maintain any assumed meaningful level of immunity against the 
targeted antigens, and even in the context of prophylactic vaccinations against infective 
agents with assumed very potent none self-antigens it is usually not possible to achieve 
efficacious long-term immunity without booster doses. These general phenomena linked to 
vaccination strategies indicate that it should also for EO2040 enhanced immunity against the 
targeted antigens be a possibility to influence efficacy by stopping booster doses. Thus, a 
meaningful safety action for the applied therapeutic vaccination strategy is to stop EO2040 
administrations at non-acceptable toxicity.       
In this trial, as in the early development of EO2401, subcutaneous (SC) administration of 
EO2040 will be applied based on the general notion that this is a well-accepted, and clinically 
easy and reproducible way for vaccine administration.  
The peptide dose utilized in non-clinical testing of the components of EO2040 was 100 µg 
per peptide. However, given that there is no predefined conversion factor to enable 
extrapolation from a safe dose in animals to a potentially safe starting dose in humans for 
this type of cancer vaccine product (as referred in the US FDA guidance for industry 
“Clinical Considerations for Therapeutic Cancer Vaccines”, October 2011 [92]), the clinical 
dose for the first cohorts in both EOGBM1-18 and EOADR1-19 was chosen based on 
reported clinical study results with other peptides for which immune responses and efficacy 
were observed after injection of 300 µg per peptide (see the current version of the EO2040 
IB).  
Immunogenicity data to support the use of the UCP2 helper peptide, combined with the 
microbiome-derived peptides, together with Montanide ISA 51 VG Sterile as adjuvant have 
been generated in vivo in HLA-A2 transgenic mice. The presence of Montanide ISA 51 VG 
supports the induction of an immune response in mice and the addition of a Th1 helper 
peptide as UCP2 showed an enhancing effect on T cell response induced by the microbiome-
derived peptide EO2315 against itself and the corresponding human TAA (see the current 
version of the EO2040 IB). 
The dosage regimen for EO2401 including a priming schedule was chosen to induce an 
efficient immune response (every 2 weeks, 4 times), and to avoid exhaustion by a 4-week 
break thereafter. The priming period is followed by a boosting period with injections every 
4 weeks to maintain an adequate immune response. The prime-boost approach was based on 
general immunization concepts, especially from the field of therapeutic cancer vaccines.  
The clinical data generated for EO2401 supports the dose and schedule which is 
implemented in the current trial for EO2040 (see Section 1.3.6.1).  
Nivolumab route of administration, dosage, dosage regimen, and treatment periods are 
aligned with current labeling for the compound [90, 91]. Of note, the interval between 
nivolumab administrations in the current trial is adjusted according to the administrations of 


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EO2040, i.e. with every 2 weeks administrations 4 times followed by every 4 weeks during 
the boosting period of EO2040 when given from the start of study treatment. The nivolumab 
schedule of administration was selected based on the wish to minimize the number of 
necessary patient visits. 
The optimal duration of immunotherapy in general is an important question without any 
current definitive answer. Likewise, and even to a greater extent in the situation of ctDNA 
defined MRD in patients with curatively treated CRC, there is no obvious guidance from 
prior trials which treatment duration would be optimal. From an immunogenicity standpoint 
data generated in the development of EO2401 indicate that a very fast expansion, after 1-2 
administrations, of T cells with killing ability of cells expressing the targeted TAAs is to be 
expected for EO2040; as far as known today, as long as boosting is continued the immune 
response at a high level will also be maintained (for EO2401, per March 2022, the longest 
maintained immune response has a duration of 44 weeks, and is ongoing - with at this time 
approx. 3% BIRC5 specific CD8+ T cells in the peripheral blood of the patient). 
Thus, it is known that a fast and prolonged immune response against the targeted TAAs can 
be achieved with the components of EO2040. However, since it is not known if this immune 
response can irradicate ctDNA defined MRD in patients with CRC, it seems reasonable to 
define a finite treatment duration for a first exploratory trial. The selected treatment duration 
is 6 months, based on the ease of timing with standard of care procedures as further ctDNA 
testing and radiographic imagining.  
Stopping study treatment early at confirmed progression, or individual patient safety 
concerns is considered standard oncology practice.  
The safety profile of the components included in EO2040 (administered with the adjuvant 
Montanide) in combination with nivolumab shown in the early clinical development of 
EO2401, was as expected based on the components of treatment, and considered supportive 
for a limited size phase 2 trial in patients with stage II-IV colorectal cancer with ctDNA 
defined MRD after completion of curative therapy. 
In addition, the early development of EO2401 indicated the potential of efficacy of the 
included vaccine components in combination with nivolumab, in established advanced solid 
tumors as glioblastoma and adrenocortical carcinoma (the latter in a population of patients 
where the majority have liver metastases, most often combined with other metastatic sites, 
including lung). 
Based on all above, the Sponsor assessment is that there is a positive benefit/risk ratio for 
performing the current trial as outlined in protocol EOCRC1-22. 
 
 


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1.4 
History of amendments 
EOCRC1-22 protocol 
version (date) 
Key points of protocol version 
(only substantial changes listed) 
  
Version 1.0 
(2022-MAR-28)  
For authority submissions 
Version 2.0 
(2002-JUL-04) 
Updated version following IND review by the FDA 
 
 


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2 
RATIONALE 
2.1 
Rationale for the study 
Detectable ctDNA after completion of curative therapies can identify patients with high-risk 
CRC, but still with a very limited tumor burden. Thus, a minimal residual disease (MRD) 
setting which can be assumed to be advantageous, especially for some treatment options, 
e.g. immunotherapy approaches. The MRD setting has, since a longer time, been targeted 
for curative treatment approaches with some success in hematological malignancies.  
Patients with ctDNA defined MRD of CRC after completion of curative therapies are highly 
likely to recur with overt metastatic disease (positive predictive value of ctDNA is 
approximately 100% by 3 years).  
Patients with ctDNA positive testing have disease shedding tumor DNA into the circulation, 
which at early stages might represent radiographically undetectable micrometastatic disease.  
The lead-time of ctDNA positivity to radiographic recurrence is up to 6-9 months that 
provides a window for early initiation of systemic therapy towards eradication of 
micrometastatic disease [22]. Clearance of ctDNA may be used as a surrogate endpoint 
under the assumption that this is necessary (albeit insufficient as of now) for eventual cure 
[93, 94]. 
We hypothesize that EO2040 in combination with nivolumab in patients with ctDNA-
defined MRD will eradicate micrometastatic disease resulting in ctDNA clearance and 
improvement in disease free survival (DFS) compared to MRD-positive historical controls. 
The background for this hypothesis has been outlined in Section 1.3. 
Patients with CRC being treated with curative intent, dependent on institution and country 
of treatment, might routinely have ctDNA analysis at key time points including at diagnosis, 
completion of each line of therapy, post-surgery, post-adjuvant therapy, and during 
surveillance. Screening for ctDNA-defined MRD is assumed to be done as standard-of-care 
per current practices. 
 
 


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3 
STUDY OBJECTIVES AND ENDPOINTS 
3.1 
Objectives  
3.1.1 
Primary objective  
 The primary objective of this trial is to assess the 6-month ctDNA clearance rate at 
therapy with EO2040 in combination with nivolumab, in patients with ctDNA-defined 
MRD of stage II-IV colorectal cancer after completion of curative therapy. 
and in sequence, provided the first objective has a positive outcome per Section 9: 
 The primary objective is, to assess the 6-month ctDNA clearance rate at therapy with 
EO2040 monotherapy, in patients with ctDNA-defined MRD of stage II-IV colorectal 
cancer after completion of curative therapy. 
o In case the first objective does not have a positive outcome per Section 9, a 
protocol amendment to adjust the second part of the primary objective to utilize 
an alternative therapeutic vaccine than EO2040 will be considered.  
ctDNA clearance is utilized as a surrogate endpoint for eventual cure, and thereby 
prolongation of disease-free survival (DFS) for patients achieving clearance.  
Note, clearance of ctDNA is characterized by the disappearance of all somatic mutations 
identified in the blood, as well as no appearance of any additional new somatic mutations, 
and radiographic investigation(s) showing no evidence of colorectal cancer (see Section 
7.8). 
3.1.2 
Secondary objectives 
To assess the following items at therapy with EO2040 in combination with nivolumab, and 
when applicable EO2040 monotherapy, in patients with ctDNA-defined MRD of stage II-
IV colorectal cancer after completion of curative therapy: 
1. safety and tolerability of study treatments,  
2. the 3-month ctDNA clearance rate, 
3. progression of colorectal cancer and death as disease-free survival (DFS), 
4. overall survival (OS), 
5. survival at 36 months after start of study therapy, and 
6. induction/expansion of T cells specific for EO2040, the components of EO2040, 
and the targeted nominal TAAs (BIRC5 and FOXM1). 
3.1.3 
Exploratory objectives 
1. To assess expression in tumor tissue (which might be from the primary diagnosis/surgery 
of CRC, or a later timepoint before study start; or samples from surgeries necessary 
according to standard of care principles during study participation) of TAAs and immune 
response/resistance related markers.  
2. To assess shifts in peripheral blood, serum, and tumor tissue (if tissue would also become 
available during study treatment) cell population/marker expressions as for instance 
specific T cell populations, T regulatory cells, myeloid-derived suppressor cells 
(MDSCs), and ctDNA. 


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3. To assess correlations between immunogenicity of EO2040, the components of EO2040, 
and the targeted nominal TAAs (BIRC5 and FOXM1) and efficacy, and safety, outcome 
parameters. 
4. To characterize T cell receptor (TCR)-variation, and possible correlations with efficacy 
outcome parameters.  
5. To assess level of tumor mutational burden in individual patients, and possible 
correlations with efficacy outcome parameters.  
6. To assess disease specific somatic and germline mutations in individual patients related 
to CRC in tumor samples and germline mutations in blood samples, and possible 
correlations with efficacy outcome parameters. 
7. To assess metagenomics on stool samples to understand the impact of study treatments 
on the microbiome, and possible influence of the microbiome composition of patients on 
safety and efficacy parameters. 
8. To characterize factors in serum, tumor samples, or blood samples, impacting antitumor 
immunity and definition of individual patient tumor inflammatory signatures, potentially 
delineating patients with a higher likelihood of tumor response to treatment (i.e. 
characterization of factors which may predict clinical benefit).  
9. To characterize changes in ctDNA allele fractions over time with therapy. 
3.2 
Endpoints 
3.2.1 
Primary endpoint 
Response to therapy will be measured with a standard-of-care, CLIA certified ctDNA assay, 
or similar certification outside of the USA, at the evaluation times specified in Section 7.1.  
Identification of somatic mutations in the blood will be confirmed by sequencing of the 
resected tumor using a CLIA-certified next-generation sequencing panel. Clearance of 
ctDNA will be characterized by the disappearance of all somatic mutations identified in the 
blood, as well as no appearance of any additional new somatic mutations.  
The primary endpoint is response to treatment at 6 months (see Section 3.1.1), which is 
defined as having clearance of ctDNA, AND not having any radiographic evidence of 
recurrence at 6 months (see also Section 7.8 for rules of assessment of an individual patient 
best overall response).  
If at least 4 out of 17 patients in the Full Analysis Set (FAS; patients who received at least 
one dose of EO2040 and for whom no important protocol deviations occurred) show 
response to study therapy, the therapy will be declared successful. 
3.2.2 
Secondary endpoints 
1. The safety and tolerability of study treatments will be determined by a descriptive 
medical assessment of the combined profile of incidences of adverse events (AEs), 
treatment-emergent AEs (TEAEs), serious AEs (SAEs), deaths, reasons for treatment 
discontinuation/delays, and laboratory abnormalities using the NCI-CTCAE v5.0 
grading system [100]. See further Section 7.7. 
2. Response to therapy at 3 months, defined as clearance of ctDNA, AND no radiographic 
evidence of recurrence (determination method will be the same as for the primary 
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3. Disease-free survival (DFS) defined as the time from start of study treatment to the date 
of first documented CRC recurrence or death due to any cause, whichever occurs first. 
Patients alive without recurrence will be censored at the date of last follow-up time. 
Recurrence is defined by the appearance of one or more new CRC lesions on imaging 
and/or other clinical evaluation such as endoscopy. 
4. Overall survival (OS), measured as the time from start of study treatment until death 
from any cause. Patients alive will be censored at the last time documented to be alive.  
5. Survival at 36 months after start of study treatment, estimated via Kaplan-Meier 
estimates. 
6. Immunogenicity and cross reactivity: immunogenicity determined as expansion of 
specific T cells comparing samples taken at baseline versus on treatment in an individual 
patient determining if the patient has a positive response to the immunization, or not, in 
relation to EO2317, EO2318, and UCP2 that compose EO2040 by interferon-gamma 
(IFN-γ) enzyme-linked immunospot (ELISpot), intracellular cytokines staining, and 
multimers staining assays. Cross reactivities with the TAAs BIRC5/survivin and 
FOXM1 will also be evaluated by the same methods. Note, cell availability at each 
sampling time sets the limit for tests possible to do, and the priority order is multimer 
staining and ELISpot firsthand, and then if possible intracellular cytokine staining (the 
latter might also intentionally not be done in all patients).  
3.2.3 
Exploratory endpoints 
1. Expression by immunohistochemistry (IHC) in tumor (and normal, if available) tissue of 
BIRC5/survivin, and FOXM1, and immune response/resistance related markers (e.g. 
HLA-A2, PD-L1, CD3, CD4, CD8, granzyme, Ki67; additional markers might be added, 
for instance if immune-resistance mechanisms relevant for immunization approaches 
would become known during trial conduct), aiming at defining immune profiles of 
tumor-infiltrating lymphocytes, and expression of immune markers on tumor cells and 
in the tumor microenvironment via determination of percentages of positive cells for the 
different markers. In addition, and as possible by available material, molecular markers 
(presence, expression levels) will also be determined, including but not limited to 
mutations, deletions, and/or amplifications, and necessary assessments for determination 
of cancer molecular subtypes. 
2. Changes between baseline and on treatment evaluations of peripheral blood, serum, and 
tumor tissue cell populations/markers as specific T cell populations (by e.g. TCR 
sequence analyses), T regulatory cells, and myeloid-derived suppressor cells (MDSCs), 
changes in immune related markers as PD-L1/L2 expression, and changes in ctDNA 
during the study. 
3. Correlations between immunogenicity (as measured by specific T cell responses, 
categorizing patients as negative, positive at baseline, or positive during treatment for 
the respective assays) of EO2317, EO2318, and UCP2 and clinical efficacy (per primary 
and secondary efficacy endpoints) and safety (TEAEs of defined specificities and grades) 
outcome parameters. Cross reactivities shown for the TAAs BIRC5/survivin, and 
FOXM1 will also be explored in the same way. 
4. Characterization of T cell receptor (TCR)-variation by sequencing, and possible 
correlations with treatment outcomes (per primary and secondary efficacy endpoints).  
5. Level of tumor mutational burden in individual patients as measured by the total number 
of nonsynonymous mutations per coding area with whole exome sequencing of tumor 


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samples, and statistical assessments of correlations of these levels with treatment 
efficacy outcomes (per primary and secondary efficacy endpoints).   
6. Disease specific somatic and germline mutations in individual patients related to CRC 
as measured by detection of nonsynonymous mutations in tumor samples and germline 
mutations in blood samples by exome sequencing, and correlations with treatment 
efficacy outcomes (per primary and secondary efficacy endpoints).   
7. Overall fecal microbiome diversity (microbial DNA sequencing for metagenomics) at 
baseline compared with on treatment; changes in relation to safety (TEAEs of defined 
specificities and grades) and efficacy outcome (per primary and secondary efficacy 
endpoints) parameters.  
8. Characterization of factors impacting antitumor immunity and definition of individual 
patient tumor inflammatory signatures, potentially delineating patients with a higher 
likelihood of tumor response to treatment. These may include measurements in serum of 
cytokines such as IL-6 and inflammatory markers such as C-reactive protein, whole 
exome sequencing of tumors, and single cell RNA-sequencing of PBMCs. 
9. Characterization of changes in ctDNA allele fractions over time with therapy. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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4 
STUDY DESIGN 
4.1 
Overall trial design and timelines 
The trial is a multi-center, open-label, non-comparative, two sequential cohort, phase 2 trial, 
to investigate as the first cohort efficacy of the microbiome-derived therapeutic vaccine 
EO2040 in combination with nivolumab in patients with stage II-IV colorectal cancer with 
ctDNA defined MRD after completion of curative therapy. Assuming a positive outcome of 
the first cohort, a second cohort assessing efficacy of EO2040 monotherapy, with the option 
of addition of nivolumab after 3 months in case of no ctDNA clearance, is planned.  
The objectives of the trial are outlined in Section 3.1. 
The study is assumed to recruit a total of 17 patients in Cohort 1, and if cohort 1 has a positive 
outcome (see Section 9), a total of 17 patients will also be recruited to Cohort 2A.  
The recruitment period is estimated to be 12 months for Cohort 1, and the same for Cohort 
2A. Thus, in total a possible recruitment time of 24 months. The planned maximum follow-
up is 36 months for an individual patient, leading to a total study duration of approximately 
6 years (adding the maximum possible needed 6 months follow-up after recruitment of the 
last patient to Cohort 1 before there is a result to advice on start/no-start of recruitment to 
Cohort 2A, and a conservative 6 months for post-last patient visit activities).   
For details regarding sample size determination and statistical analyses see Section 9. 
Figure 15 gives a schematic overview of the trial design, and an overview of the treatment 
schedule is displayed in Figure 16 (for further specifics see the Study Schedule in Section 
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Figure 15 : EOCRC1-22 Overview trial design 
 
 
Figure 16 : EOCRC1-22 treatment schedule, scan and ctDNA assessments, and blood 
sampling for immune testing and serum collection  
 


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4.2 
General trial design 
There will be a 2-stage consent procedure for the trial, including a first consent for basic 
disease, age information, and HLA-typing (or the use of already available information in the 
patients' file; such information even if based on testing before the screening period is 
acceptable) since a large proportion of the patients (approximately half of the patients) will 
not match the HLA-A2 prerequisite to receive the HLA-A2 specific EO2040 immunization 
compound. The reason for the initial minimized consent (related to the procedure of  
HLA-testing, and before testing also establishing based on available information that the 
patient is eligible in relation to CRC type, prior therapy, and has an age ≥ 18 years) is to 
spare the necessity of all other testing for possible enrollment in the trial for patients who 
are not known to be HLA-A2 positive.  
The second stage of the consent procedure will be related to treatment, and follow-up aspects 
of the trial procedures.  
Patients who have provided informed consent per above, and who meet all the inclusion 
criteria, and do not meet any of the exclusion criteria, can be finally evaluated by the 
Sponsors Medical Monitor for start study treatment. The recruiting site should submit a start 
of treatment request (after also having filled out all applicable information in the eCRF) to 
the Medical Monitor at least 24 hours before anticipated study treatment start (procedures 
are described in a separate Cohort Administration Charter); the Medical Monitor will assess 
the request within 12 hours of receipt.   
Each patient will individually participate in the study for a maximum of approximately 3.5 
years, including approximately 1 month for screening, 6 months for study treatment, and a 
maximum of 3 years follow-up after start of study treatment. 
Study treatments will be administered until study treatment termination criteria as outlined 
in Section 5.3.3 are fulfilled, e.g. reaching maximum study treatment period (6 months), 
unacceptable toxicity, or relapsing disease. At the time of stopping study treatment, 
appropriate other treatment will be advised by the treating physician on an individual patient 
basis. The patient should continue, even if study treatment is stopped, study follow-up 
measures for as long as the individual patient consent for follow-up is not withdrawn, the 
site is open, and the study not terminated per plan, or early, but for a maximum length of 3 
years after start of study treatment. 
All assessments of possible relapse in the trial will be determined at the sites by the principal 
investigators, or designees, according to the standard clinical criteria used at the site. 
Of note, there is no current knowledge publicly available regarding the potential of 
pseudoprogression at immunotherapy in patients with ctDNA defined MRD of CRC. Thus, 
at scanning, especially at the 3-month study timepoint, if a relapse is indicated by scanning 
it is recommended to, if at all possible, also document the relapse with histology or 
cytology. 
4.3 
Trial Safety  
The safety oversight of the trial is a combined effort of the investigators with their local 
study teams, the Independent Data Monitoring Committee (IDMC), and the Sponsor (also 
represented when applicable by designated CROs). The setup and tasks of the IDMC are 
outlined below (see Section 8.6), and details of the IDMC processes and procedures, are also 
outlined in a separate charter. 


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Pre-planned safety monitoring measures are outlined in the Study Schedule (see Section 7.1).  
Safety profiles of the study treatments are outlined in Section 1.3.2 (EO2040), and Section 
6.2.5 (nivolumab). 
Administration of EO2040 in combination with nivolumab is described in Section 6.3, and 
treatment modifications are described in Section 6.4. 
For all patients during the whole trial treatment, the following safety rules apply: 
 All related Grade ≥ 3 adverse events should be reported within 24 hours to the Sponsor 
via the Medical Monitor (reporting can be via the medical monitoring e-mail 
[medicalmonitoring-crc@enterome.com] or the medical monitoring telephone [+33 
(0) 6 14 31 07 59]) and be reported as an SAE if it meets such criteria (see Section 
8.5.3). Note, if the eCRF is filled out with the related Grade ≥ 3 adverse event within 
24 hours this is also accepted as information to the Sponsor since there will be a special 
alert system implemented for such recordings in the eCRF.  
 
 In case a related Grade 5 AE would be observed, recruitment to the trial will be held until 
a decision is made by the Sponsor following an IDMC recommendation regarding further 
study conduct actions (during the period of assessment and decision making, other 
already started patients are allowed to continue treatment if no other recommendation is 
provided by the IDMC and/or decided by the Sponsor). 
 In case a related clinical Grade 4 AE would be observed, all components of study 
treatment will be permanently stopped in the individual patient experiencing the event, 
and new patient trial treatments will be held until a decision is made by the Sponsor 
following an IDMC recommendation regarding further study conduct actions (during the 
period of assessment and decision making, other already started patients are allowed to 
continue treatment if no other recommendation is provided by the IDMC and/or decided 
by the Sponsor). 
 In case a related clinical Grade 3 AE would be observed, the Investigator will temporarily 
hold the administration of all components of study treatment in the individual patient 
experiencing the event until a thorough assessment of the case has been made. A decision 
to restart the study treatment in the individual patient experiencing the event can be made 
by the Investigator responsible for the patient and the Sponsor in collaboration, with the 
option for any of them to defer the assessment for recommendation of a decision to the 
IDMC. Other patients in the trial will continue their study treatments as planned, if not 
otherwise recommended by the IDMC and/or decided by the Sponsor. 
 In case of immune-related adverse events related to study treatment instructions for 
treatment modifications as outlined in Section 6.4.1 should be followed. Section 12.5 
also includes management algorithms regarding nivolumab in case of specific adverse 
reactions. 
 See Section 12.4 regarding precautions in relation to possible infusion related reactions, 
and treatment of such reactions. 
 In addition, any Investigator in the trial or the Sponsor can at the detection of a medically 
important event call upon the IDMC to assess the event and make a recommendation 
regarding further study conduct actions to the Sponsor for decision. 
 Note: Clinical AEs like, for instance, alopecia and transient laboratory abnormalities 
without clinical significance (e.g. no need for medical actions or treatments) will not be 


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considered for actions and decisions per above. The clinical significance of such clinical 
AEs and laboratory abnormalities will be assessed on a case-by-case basis by the 
Investigator in collaboration with the Medical Monitor of the Sponsor, and when deemed 
appropriate in consultation with the IDMC. All decisions by Investigator/Sponsor to 
consider a related ≥ Grade 3 clinical AE or transient laboratory abnormality to be non-
clinically significant per above will be documented and the documentation provided in 
writing to the IDMC, as soon as the assessment by the Investigator and Sponsor is 
finalized; it will be assessed by the individual IDMC members when received, and it is 
to be discussed by the IDMC at their next scheduled meeting. 
4.4 
End of study 
4.4.1 
Planned study completion and study duration 
The clinical data cut-off for the final study analysis, assuming a positive outcome of Cohort 
1 and recruitment to Cohort 2A, is planned to be 36 months after the last patient has been 
starting study treatment in Cohort 2A (or if this patient is not alive, when the next "last started 
patient" has been followed for 36 months after the start of study therapy).  
The planned end of study, defined as the date of the last visit of the last patient in the study, 
is based on the foreseen clinical data cut-off per above.  
The assumed recruitment time is 12 months per cohort including 17 patients, and with the 
above outlined minimum follow-up of 36 months (from start of study treatment) for an 
individual patient at the time of the final analysis, the trial duration from start of recruitment 
until last patient visit for the final analysis will be approximately 6 years (adding the 
maximum possible needed 6 months follow-up after recruitment of the last patient to Cohort 
1 before there is a result to advice on start/no-start of recruitment to Cohort 2A, and a 
conservative 6 months for post-last patient visit activities; e.g. data cleaning including query 
resolution, document collection, etc.).    
Study sites will be closed upon decision (per plan or earlier) and once all patients at the site 
have completed the study (in line with decision to close study) and when all close out 
activities have been finalized. A study site is considered closed when all required documents 
and study supplies have been collected and a study site closure visit has been performed, 
except for the sites without any patients included which could be administratively closed by 
a close-out letter. 
4.4.2 
Early site closure and early study termination 
The Sponsor reserves the right to close a study site or terminate the study at any time for any 
reason at the sole discretion of the Sponsor.  
Reasons for early study termination may include, but are not limited to: 
 safety findings unknown to date (i.e. not previously reported in any similar 
investigational study drug trial with respect to their nature, severity, and/or duration), or 
significantly changed frequency, severity, or duration of known/anticipated/previously 
reported safety events, 
 medical or ethical reasons affecting the continued performance of the study, 
 difficulties in recruitment of patients,  
 regulatory authority request, and 


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 cancellation of development of EO2040. 
The Investigator may initiate study site closure at any time, provided there is reasonable 
cause, and adequate notice is given to the Sponsor in advance of the intended site closure.  
Reasons for the early closure of a study site by the Sponsor may include, but are not limited 
to: 
 failure of the Investigator to comply with the protocol, the requirements of the 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC) or local health 
authorities, the Sponsor's procedures, or Good Clinical Practice (GCP) guidelines, and 
 inadequate recruitment of participants by the Investigator. 
4.5 
Discussion of study design 
The trial is a multi-center, open-label, non-comparative, two sequential cohort, phase 2 trial, 
to investigate as the first cohort efficacy of the microbiome-derived therapeutic vaccine 
EO2040 in combination with nivolumab in patients with stage II-IV colorectal cancer with 
ctDNA defined MRD after completion of curative therapy. Assuming a positive outcome of 
the first cohort, a second cohort assessing efficacy of EO2040 monotherapy, with the option 
of addition of nivolumab after 3 months in case of no ctDNA clearance, is planned.  
The treatment setting as such, i.e. treatment of patients with CRC who after curative therapy 
have a ctDNA defined MRD, is by itself novel. However, detectable ctDNA after completion 
of curative therapies has nearly 100% positive predictive value for radiographic recurrence. 
Multiple studies demonstrate that the presence of ctDNA likely reflects radiographically 
unseen micrometastases, or MRD, which is predictive of recurrence and a useful screening 
tool for high-risk patients [21].  
The lead-time of ctDNA positivity to radiographic recurrence is up to approximately 9 
months in early-stage disease, which provides a window for evaluation of novel therapies in 
a setting for which there are no current standard-of-care guidelines [22].  
Clearance of ctDNA is in the current study used as a surrogate endpoint to assess within the 
assumed window before there is a documented radiographic recurrence if the study treatment 
can impact this disease parameter. Standard outcome parameters as DFS and OS will also 
be followed.   
The predetermined study treatment duration of 6 months is linked to the assumed window 
before documented radiographic recurrence, but also to the current knowledge regarding 
time until tumor impact in other solid tumors by a microbiome-derived therapeutic vaccine 
in combination with nivolumab (i.e. 6 months is assumed to be a long enough treatment 
duration for this specific mode of therapy to have had a relevant chance to show efficacy).  
The early exploratory nature of the trial in relation to the setting makes, in the view of the 
Sponsor, an open-label design adequate. 
The design with two sequential cohorts starting with the assumed most efficacious cohort 
first (direct combination of EO2040 with nivolumab from treatment day 1; available 
experience from other microbiome-derived therapeutic vaccine trials indicate that the 
expansion of specific T cells is faster if the combination is used upfront, instead of delaying 
the anti-PD1 blockade) has been selected to as fast as possible evaluate if the treatment 
concept might impact, at all, ctDNA positivity. If the initial cohort, with the current 
maximum concept treatment, would have a positive outcome, a modification of study 
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vaccine by itself, or with a delayed start of nivolumab, would also be efficacious. If this 
would be the case, one would potentially be able to spare patients the risk of some adverse 
events linked to nivolumab. Of note, neither the trial investigators, nor the Sponsor, 
considers it adequate to reverse the order of the cohorts since the setting argues for an 
approach directed towards maximum efficacy. Also, with the predetermined relatively short 
treatment duration it is assumed that the toxicity burden from nivolumab will be limited, and 
acceptable, considering the setting.    
The trial includes, due to the specific characteristics of the combination of EO2040 and 
nivolumab key well defined strategies for patient management at possible immune-mediated 
reactions (see Section 6.4.1 and extended treatment algorithms in Section 12.5). 
It is acknowledged that the trial is intensive regarding sampling. However, an important 
parameter for evaluation in the trial is to analyze EO2040-induced immunity and the tests 
for such analyzes unfortunately demand relatively high volumes of blood for adequate tests 
to be run (e.g. PBMC); a minimization strategy with regard to sampling has been applied, 
still without risking the appropriate possibility to get relevant readouts. The future potential 
use of EO2040 might be directed by the outcome of the analyses of induced immunity in the 
trial. 
The efficacy assessments by scanning in the trial are based on site/local investigator 
assessments which is considered adequate for fast decisions regarding study treatments, and 
in context of key endpoints related to ctDNA testing. 
 
 
 


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5 
POPULATION 
5.1 
Inclusion criteria 
To be eligible to receive study treatment, a patient must meet all the criteria below: 
1. Provided written informed consent prior to any study-related procedures (initial consent 
is for screening part #1 procedures, and a second consent is for screening part #2 
procedures; see Section 7.2 and Section 7.3, respectively). 
2. Histological confirmation of colorectal cancer (confirmation at initial diagnosis is 
sufficient). 
3. Post R0-resection (i.e. microscopically margin-negative resection and no gross or 
microscopic tumor remaining in the primary tumor bed) of stages II, III, or IV CRC and 
completion of all planned standard of care perioperative and/or adjuvant therapies. 
a. Note, if there is doubt the patient has received all relevant standard of care 
therapy, the patient should be discussed with the trial Medical Monitor, who can 
also defer the case for decision by the Global Coordinating Investigator. 
4. Presence of minimal residual disease as defined by a positive ctDNA assay after 
completion of all planned standard of care therapies. 
a. Note, patients may be identified for enrollment with any CLIA-certified, or 
similar certification outside of the USA, ctDNA assay for MRD as standard of 
care or in a surveillance trial. However, MRD status will be confirmed with the 
Signatera assay prior to initiation of study therapy (i.e. within the screening part 
#2 procedures) for all patients who have not had a Signatera research grade 
testing where all parameters from the testing can be provided for the current 
trial. 
5. Age ≥ 18 years old. 
6. Human leukocyte antigen (HLA)-A2 positive. 
7. No evidence of radiographic disease (computer tomography or magnetic resonance 
imaging; optimized to detect metastatic disease, e.g. with contrast as applicable) that 
requires immediate therapeutic intervention as assessed by the treating physician, within 
28 days of (before or after) a positive ctDNA assay.  
a. Note, if there are lesions on scanning which are highly suspicious for being 
metastatic CRC even if no immediate therapeutic intervention is needed, this is a 
clear indication to NOT enroll the patient in the trial. At doubt the case should 
be discussed with the trial Medical Monitor, who can also defer the case for 
decision by the Global Coordinating Investigator. 
b. Note, if the scanning is not done within -7 days/+28 days of the positive Signatera 
assay which is accepted as confirmatory test for the trial, the scanning needs to 
be repeated within the screening part #2 procedures. 
8. ECOG performance status 0 or 1 (see Section 12.1).  


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9. Female patients of childbearing potential must have a negative serum pregnancy test 
within 72 hours prior to start of study therapy. 
10. Considering the embryofetal toxicity of the immune checkpoint inhibitor (ICI) shown in 
animals’ models, the following recommendations for contraception must be followed: 
a. If not surgically sterile, female patients of childbearing potential age must use highly 
effective contraception from signing the Informed Consent Form (ICF) through 
6 months after the last treatment dose administered. Highly effective contraception 
includes (according to Clinical Trial Facilitation Group: Recommendations related 
to contraception and pregnancy testing in clinical trials [104]): 
i. 
combined (estrogen and progesterone containing) hormonal contraception 
associated with inhibition of ovulation: oral, intravaginal, transdermal, 
ii. 
progestogen-only hormonal contraception associated with inhibition of 
ovulation: oral, injectable, implantable,   
iii. 
intrauterine device (IUD), 
iv. 
intrauterine hormone-releasing system (IUS), 
v. 
bilateral tubal occlusion, 
vi. 
vasectomized partner, and 
vii. 
sexual abstinence when in line with the preferred and usual lifestyle of the 
patient (e.g. periodic abstinence is not considered a highly effective method). 
In each case of delayed menstrual period (over 1 month between menstruations), 
confirmation of absence of pregnancy is strongly recommended. This recommendation 
also applies to women of childbearing potential with infrequent or irregular menstrual 
cycles. 
b. If not surgically sterile, male with female partner of childbearing potential age must 
use condom from signing the ICF through 6 months after the last treatment dose 
administered. Males must ensure that their partners of childbearing potential use 
highly effective contraception also. Note, the contraception guidelines for males are 
not related to nivolumab but kept as a conservative precaution in this early 
development protocol in relation to EO2040. 
12. Patients willing and able to comply with the scheduled visits, treatment plan, laboratory 
tests, and other study procedures indicated in the protocol. 
5.2 
Exclusion criteria 
Patients who meet any of the following criteria will not be eligible to participate in the study: 
1. Patients treated with dexamethasone > 2 mg/day or equivalent (i.e., 13 mg/day of 
prednisone) within 14 days before start of study therapy, unless required to treat an 
adverse event (AE). 
Note: The criterion implies the patient should not receive treatment with dexamethasone 
> 2 mg/day or equivalent at the actual time of a screening visit (single time point 
assessment), and within 14 days before the start of study treatment (unless required to 
treat an AE); the latter part of the criterion should be checked at the time of start of 
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2. Patients treated with radiotherapy within 12 weeks, and cytotoxic chemotherapy therapy 
within 28 days (or 5 half-lives of the compound(s) administered if longer) before study 
treatment start. 
3. Patients with persistent Grade ≥ 2 toxicities (according to NCI-CTCAE v5.0). Toxicities 
must be resolved for at least 2 weeks to Grade 1 or less. However, alopecia, neuropathy, 
and other persisting toxicities not constituting a safety risk based on Investigator’s 
judgment are acceptable. 
4. Patients who have received any prior treatment with compounds targeting PD1, PD-L1, 
CTLA-4, or similar compounds where general resistance against therapeutic vaccination 
approaches might have developed.  
5. Patients with the following abnormal laboratory values: 
a. Hemoglobin < 10 g/dL (6.2 mmol/L); transfusion is acceptable to reach the value. 
b. Absolute neutrophil count decrease (<1.5 x109/L).  
c. Platelet count decrease (< 75 ×109/L). 
d. Total bilirubin > 1.5 ×upper limit of normal (ULN; according to the performing 
laboratory’s reference ranges); except participants with Gilbert Syndrome who 
must have a total bilirubin level of < 3.0 xULN.  
e. Alanine aminotransferase (ALT) > 3 ×ULN. 
f. Aspartate aminotransferase (AST) > 3 ×ULN. 
g. Serum creatinine increase (> 1.5 ×ULN). 
h. Abnormal thyroid function per local laboratory levels; note, patients with 
hypothyroidism only requiring hormone replacement therapy, and patients with 
long term (judged by the treating physician) stable antithyroid therapy due to 
hyperthyroidism, are permitted to enroll. Patients with abnormal thyroid 
laboratory values judged by the treating physician as clinically non-relevant are 
also eligible. 
6. Patients with presence of other concomitant active, invasive malignancies that may 
interfere with ctDNA analysis (known clonal hematopoiesis of unknown potential 
allowed). 
7. Patients with clinically significant active infection, cardiac disease, significant medical 
or psychiatric disease/condition that, in the opinion of the Investigator, would interfere 
with the interpretation of patient safety or study results or that would prohibit the 
understanding or rendering of informed consent (i.e. only consent able patients can be 
enrolled in the study) and compliance with the requirements of the protocol – including 
(but not limited to): 
a. Bacterial sepsis, COVID-19, or other similarly severe infections (clinical 
assessment is the basis for exclusion of severe infections; if clinical suspicion, 
adequate testing should be performed to exclude severe infections). 
b. New York Heart Association > Grade 2 congestive heart failure within 6 months 
prior to start of study therapy (see Section 12.2). 
c. Uncontrolled or significant cardiovascular disease, including: 
i. myocardial infarction within 6 months prior to start of study therapy, 


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ii. uncontrolled/unstable angina within 6 months prior to start of study 
therapy, 
iii. diagnosed or suspected congenital long QT syndrome, and 
iv. any history of clinically significant ventricular arrhythmias (such as 
ventricular tachycardia, ventricular fibrillation, or Torsades de 
pointes). 
d. Stroke within 6 months prior to start of study therapy. 
e. Concurrent neurodegenerative disease. 
f. Dementia or significantly altered mental status. 
8. Patients with suspected autoimmune or active autoimmune disorder or known history of 
an autoimmune neurologic condition (e.g., Guillain-Barré syndrome). 
Note-1, patients with a diagnosed inflammatory bowel disease as ulcerative colitis or 
Crohn’s disease, should be excluded. 
Note-2, patients with vitiligo, type I diabetes mellitus, hypothyroidism due to 
autoimmune condition only requiring hormone replacement therapy, psoriasis not 
requiring systemic therapy, or conditions not expected to recur in the absence of an 
external trigger are permitted to enroll. 
9. Patients with a history of solid organ transplantation or allogeneic hematopoietic stem 
cell transplantation. 
10. Patients with a history or known presence of tuberculosis. 
11. Pregnant and breastfeeding patients. 
12. Patients with a history or presence of human immunodeficiency virus (HIV) and/or 
active hepatitis B virus (HBV)/hepatitis C virus (HCV). 
13. Patients who have received live or attenuated vaccine therapy used for prevention of 
infectious diseases including seasonal (influenza) vaccinations within 4 weeks of the first 
dose of study drug. 
14. Patients with a history of hypersensitivity to any excipient, or active substance, present 
in the pharmaceutical forms of applicable study treatments. 
15. Patients under treatment with immunostimulatory or immunosuppressive medications, 
including herbal remedies, or herbal remedies known to potentially interfere with major 
organ function. 
16. Patients who have received treatment with any other investigational agent, or 
participation in another clinical trial (clinical trial including active interventions; 
participation in clinical trials for data collection purposes only are permitted) within 28 
days (or 5 half-lives if longer) prior to start of study therapy. Note, during participation 
in the current study, parallel participation in other interventional studies is not allowed 
(participation in clinical trials for data collection purposes only are permitted; also, in 
the follow-up parts of interventional studies).   


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5.3 
Removal of patients from therapy or study 
5.3.1 
Patient withdrawal of consent 
Patients may voluntarily withdraw from the study treatment, or the study as such, including 
procedures, at any time at his/her request for any reason.  
In instances where patient consent is withdrawn, the Investigator must clarify to which 
degree the patient consent is withdrawn, i.e. whether the patient still is willing to continue 
to participate in some study specific procedures, or not. The degree of consent withdrawal 
should be carefully documented in patient files and in study documents. 
5.3.2 
Screen failures 
Screen failures are defined as patients who consent to participate in the clinical study but are 
not subsequently treated with study drug(s) due to failure of the eligibility criteria. For 
documentation reasons all study related items performed before the determination of the 
status of screen failure should be documented in the eCRF.  
5.3.3 
Criteria for treatment discontinuation 
Patients should stop study treatment for any of the following reasons: 
 reached maximum defined study treatment period (EO2040 in combination with 
nivolumab 6 months; see specifics in Study Schedule Section 7.1), 
 intolerable toxicity in relation to study treatment and adverse events requiring treatment 
discontinuation according to this protocol (see Section 4.3 and Section 6.4, 
respectively), 
 pregnancy, 
 confirmed tumor relapse by scanning, 
 any medical condition which may jeopardize the patient’s safety if he/she continues trial 
treatment,  
 major protocol violation (including non-compliance to study schedule by the patient) 
judged to significantly impact safety of the patient or interpretability of the trial, 
 decision by the Investigator, Sponsor, or any regulatory authority, that treatment 
discontinuation is in the best interest of the patient,  
 patient withdrawal of consent to receive further study treatment, and 
 planned or early termination of the study (see Section 4.4). 
5.3.4 
Lost to follow-up 
A patient will be considered lost to follow-up if he/she repeatedly fails to return for 
scheduled visits 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: 
 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 
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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 
method). These contact attempts should be documented in the patient’s medical records. 
5.3.5 
Criteria for study participation termination  
Reasons for patient withdrawal from the study may include, but are not limited to: 
 patient withdrawal of consent for any further trial procedures (see also Section 5.3.1), 
 patient lost to follow-up, 
 major protocol violation (including non-compliance to study schedule by the patient) 
judged to significantly impact safety of the patient or interpretability of the trial, 
 decision by the Investigator, Sponsor, or any regulatory authority, that study 
participation discontinuation is in the best interest of the patient,  
 planned or early termination of the study (see Section 4.4). 
5.3.6 
Follow-up of patients discontinued from study treatment or withdrawn 
from study 
The primary reason for study treatment discontinuation should be documented in the eCRF. 
At treatment discontinuation, patients will complete follow-up visits according to Section 
7.5. The first follow-up visit is to be completed 30 days after completion of last dose of study 
treatment.  
In addition to the 30-days visit, for consistency with other nivolumab clinical trial protocols 
as advised by Bristol-Myers Squibb (marketing authorization holder for nivolumab), 100 
days visit following discontinuation of nivolumab treatment is also targeted. 
In cases of withdrawal from the study, every effort should be made to obtain adequate 
information. The primary reason for withdrawal from the study should be documented in the 
eCRF. If possible, patients will complete a follow-up visit according to Section 7.5. 
In cases with complete informed consent withdrawal (see also Section 5.3.1), patients will 
not be followed for any reason, although date of death will be recorded, where applicable 
and possible to retrieve.  
5.3.7 
Procedures for handling incorrectly enrolled patients 
If a patient is found to have been incorrectly enrolled in the study, the Sponsor must be 
notified as soon as the error is discovered. The Sponsor will decide on the appropriate action 
to take regarding study treatment and continuation of the patient in the study; if applicable 
the IDMC should be consulted. 
5.3.8 
Patient replacement 
Generally, no replacement is foreseen. However, the primary analysis for each cohort builds 
on an assessment of 17 patients in the Full Analysis Set (FAS; patients who received at least 
one dose of EO2040 and for whom no important protocol deviations occurred). Thus, if there 
would be relevant important protocol deviation(s) in a patient impacting the possibility to 
assess the primary endpoint the patient might be replaced based on a decision by the Sponsor 
after consultation with the IDMC. 
 
 


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6 
TREATMENT OF PATIENTS 
6.1 
Description of EO2040 
6.1.1 
Pharmaceutical form 
The Investigational Medicinal Product (IMP) of the study, EO2040 Drug Product (DP), 
consists of a water/DMSO solution (67/33 v/v) containing 3 peptide drug substances 
(EO2317, EO2318, and UCP2). The EO2040 DP is then extemporaneously emulsified with 
the adjuvant Montanide ISA 51 VG to form a water-in-oil emulsion for subcutaneous 
injection. The final concentration of each peptide in the emulsified DP in adjuvant is 300 
µg/mL. 
Further details regarding the components of EO2040 can be found in Section 1.3, and in the 
current version of the EO2040 IB. 
6.1.2 
Preparation of the emulsion 
The IMP is provided to the clinical sites in a form of a mixing kit containing 2 vials (1 
EO2040 DP vial and 1 adjuvant vial, i.e., Montanide ISA 51 VG) plus additional 
reconstitution material. The Investigator or pharmacist at the investigational site will 
undertake specific training on handling and preparing study treatment and will ensure that 
Good Pharmacy Practices are followed during the preparation and reconstitution process of 
EO2040. 
Detailed composition of EO2040 is presented in Table 5. 
Detailed instructions for the reconstitution of emulsified EO2040 DP in adjuvant, i.e. 
Montanide ISA 51 VG, will be provided in the Pharmacy Manual. 
Table 5 : Composition of the final emulsified EO2040 DP in adjuvant obtained by 
reconstitution 
 
 
 
 
     
 
*Referring to the volume of 600 μL used for reconstitution. 
No. 
Code 
Component vial 
Function in 
formulation 
Amount 
Total* 
Per 1 mL 
(SC single 
dose) 
1 
EO2317 
EO2040 DP vial 
Drug substance 
0.36 mg 
0.30 mg 
2 
EO2318 
Drug substance 
0.36 mg 
0.30 mg 
3 
UCP2 
Drug substance 
0.36 mg 
0.30 mg 
4 
Water for 
injection 
Solvent 
0.40 mL 
0.34 mL 
5 
Dimethyl 
sulfoxide 
Solvent 
0.20 mL 
0.16 mL 
6 
Montanide 
ISA 51 VG 
Sterile 
Adjuvant vial 
Adjuvanting 
solution 
0.60 mL 
0.50 mL 


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 EO2040 vial: (a sterile solution in a glass vial) comprises EO2040 DP, the 3-peptide 
mixture solution, each at 600 µg/mL in DMSO and water for injection, intended for 
proprietary use in this clinical trial with EO2040. It contains an extractable volume 
of not less than 0.6 mL solution for the reconstitution process to obtain the final IMP 
EO2040 DP emulsified in adjuvant. 
 Adjuvant vial: (a sterile solution in a glass vial) comprises the adjuvant Montanide 
ISA 51 VG which is commercially available. 
 Regarding additional material see Section 6.1.3. 
Reconstitution process 
The final emulsified EO2040 DP in adjuvant will be reconstituted by the investigational site 
pharmacy, following the Pharmacy Manual and the related instructions for reconstitution for 
the emulsified EO2040 DP in adjuvant Montanide ISA 51 VG.  
The final emulsified EO2040 DP in adjuvant will be obtained by emulsifying 600 μL of the 
EO2040 DP, the peptide mixture solution, with 600 μL of Montanide ISA 51 VG solution 
using a 2-syringe mixing process. In the starting sub-cohort 1a, 1 mL of the resulting 
emulsion of EO2040 will be administered as an SC injection within 4 hours from the start 
of the reconstitution (i.e. from the start of mixing EO2040 and Montanide ISA 51 VG). This 
administration volume will contain a dose of 300 μg of each of the 3 peptides, corresponding 
to a total peptide drug substance dose of 0.9 mg, accordingly. The composition of the final 
emulsified EO2040 DP in adjuvant obtained by reconstitution is provided in Table 5. 
Training on the reconstitution process will be given. 
Deliver the final emulsion cautiously, e.g. do not deliver/transport the final emulsion via 
pneumatic tube systems, to avoid splitting the drug emulsion. 
6.1.3 
Packaging and labelling  
EO2040 will be manufactured as per Good Manufacturing Practice (GMP) and supplied by 
the Sponsor as a mixing kit composed of: 
 1 EO2040 DP vial which is a sterile 2R Type I glass vial containing 0.750 mL of 
EO2040 DP, 
 1 adjuvant vial (i.e., Montanide ISA 51 VG, 3 mL vial), and 
 ancillary material required for the emulsion preparation and injection (i.e., syringes, 
needles, connector, syringe tip cap). 
The mixing kit is supplied as a single box presentation. 
EO2040 DP vial will be labeled with a study-specific primary label. Labels with 
requirements for the specific country will also be applied. The adjuvant vial (Montanide ISA 
51 VG) will be labeled with the original primary label of commercial supplier. The 
secondary packaging (i.e. carton box) will be labeled with a study-specific secondary label, 
which will state storage conditions. Label text will be approved by the Sponsor and include 
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For all vials, a system in accordance with GMP will be used; ensuring each dose of EO2040 
can be traced back to the respective bulk ware of the ingredients. A complete record of batch 
numbers and expiry dates of all study drugs and labels will be maintained in the Sponsor’s 
study file. 
A detailed process for ordering the IMP, handling and administration, packaging, and 
labeling the IMP will be specified in the Pharmacy Manual. 
6.1.4 
Storage and stability 
The study treatment must be stored in a secure limited access area according to local 
regulations. The study treatment is presented as a single box mixing kit that is stored at 5 ± 
3°C. 
6.2 
Description of nivolumab 
6.2.1 
Pharmaceutical form 
Nivolumab is sterile concentrate solution for infusion administration. The solution is clear 
to opalescent, colorless to pale yellow liquid that may contain a few light particles. The 
solution has a pH of approximately 6.0 and an osmolality of approximately 340 mOsm/kg. 
List of excipients include; sodium citrate dihydrate, sodium chloride, mannitol (E421), 
pentetic acid (diethylenetriaminepentaacetic acid), polysorbate 80, sodium hydroxide (for 
pH adjustment), hydrochloric acid (for pH adjustment), water for injections. 
Further details regarding nivolumab can be found in the European SmPC or the US PI for 
nivolumab [90, 91]. 
6.2.2 
Preparation of the formulation 
Instructions for preparation of nivolumab for infusion can be found in the European SmPC 
or the US PI for nivolumab [90, 91]. 
6.2.3 
Packaging and labeling  
Nivolumab will be obtained from commercial sources by the Sponsor and clearly labeled for 
use within the trial. 
Adequate supplies of nivolumab in 24 mL glass vials will be provided to each investigational 
site for preparation and infusion. The vial stopper will be a coated butyl rubber with a grey 
aluminum flip-off seal. 
6.2.4 
Storage and stability 
Nivolumab should be stored in the refrigerator at 5 ± 3°C in its original package in order to 
protect from light. Once opened, the medicinal product should be infused or diluted, and 
infused immediately (99, 100).  
If not used immediately, chemical and physical in-use stability of nivolumab has been 
demonstrated for 24 hours at 5 ± 3°C protected from light and a maximum of 8 hours at 20°C 


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to 25°C with room light (this 8-hour period of the total 24 hours should be inclusive of the 
product administration period). 
6.2.5 
Expected safety profile of nivolumab 
The following summary is based on the European SmPC [91]. 
In the pooled dataset of nivolumab as monotherapy across tumor types (n = 3771) with 
minimum follow-up ranging from 2.3 to 28 months, the most frequent adverse reactions (≥ 
10%) were fatigue (46%), musculoskeletal pain (31%), diarrhea (26%), nausea (24%), cough 
(24%), rash (24%), dyspnea (18%), pruritus (18%), decreased appetite (18%), constipation 
(17%), abdominal pain (16%), upper respiratory tract infection (16%), arthralgia (15%), 
pyrexia (14%), vomiting (14%), headache (13%) and oedema (11%). The majority of 
adverse reactions were mild to moderate (Grade 1 or 2). With a minimum of 63 months 
follow-up in NSCLC, no new safety signals were identified. 
In addition, nivolumab is associated with immune–related adverse reactions. With 
appropriate medical therapy, immune-related adverse reactions resolved in most cases. 
Table 6 presents the percentage for immune-related adverse reactions leading to permanent 
discontinuation of nivolumab and the percentages requiring high-dose corticosteroid dosing. 
For the laboratory abnormalities observed with nivolumab monotherapy, please refer to the 
European SmPC or to the US PI [90, 91]. 
Of the 3224 patients who were treated with nivolumab monotherapy 3 mg/kg or 240 mg 
every 2 weeks and evaluable for the presence of anti-product-antibodies, 286 patients (8.9%) 
tested positive for treatment-emergent anti-product-antibodies with sixteen patients (0.5%) 
testing positive for neutralizing antibodies. 
Generally, no overall differences in safety regarding nivolumab were reported between 
elderly (≥ 65 years) and younger patients (< 65 years). In the nivolumab non-squamous non-
small cell lung cancer study (CA209057), the safety profile in patients with baseline renal 
or hepatic impairment was comparable to that in the overall population. These results should 
be interpreted with caution due to the small sample size within the subgroups. 


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Table 6 : Nivolumab immune-related adverse reactions leading to permanent 
discontinuation or requiring high-dose corticosteroids 
 
a: At least 40-mg daily prednisone equivalent. 
b: Frequency is based on the number of patients who experienced the immune-related adverse reaction. 
6.3 
Administration of EO2040 and the combination of EO2040 and nivolumab 
Note, detailed instructions are also specified in the Pharmacy Manual. 
 EO2040 should be injected within 4 hours from the start of the reconstitution (i.e. from 
the start of mixing EO2040 and Montanide ISA 51 VG). 
 EO2040 (1 mL of the emulsion) should be administered subcutaneously (SC) in a 
rotating way by injection (thus, an injection site will be used every fourth time), with the 
injection sites being right and left upper extremity and right and left lower 
extremity/inguinal area, with the more specific locations being: 


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o For upper extremity = SC injection in the area of armpit, or deltoid muscle of 
arm, depending on the constitution of the patient. 
o For lower extremity = SC injection in the area of groin, or anterolateral thigh 
muscle, depending on the constitution of the patient. 
 EO2040 will during the initial 6 weeks be administered SC 3 times, each administration 
followed by a 2-week observation period. After the initial 6 weeks EO2040 will be 
administered with a 4 weekly interval starting from the administration at week 7. See 
also Study Schedule in Section 7.1. 
 All patients should be monitored for 1 hour after an EO2040 administration.  
 Dose modifications for EO2040 include withholding of doses and treatment 
discontinuation (see Section 6.4).  
 At combination treatment when EO2040 and nivolumab are to be administered on the 
same day, the order of administration is to start with EO2040, followed by a 1-hour 
observation period, after which the nivolumab infusion should be administered 
intravenously (IV) according to local guidelines. The dose of nivolumab should be 240 
mg at visit 1 (week 1), visit 2 (week 3), and visit 3 (week 5), and from visit 4 (week 7) 
and onwards 480 mg per administration every 4 weeks. Observation after nivolumab 
administration should follow local guidelines. 
 The only adjustments regarding dose for nivolumab are withholding of one or several 
doses, or discontinuation of treatment; such measures should be taken in accordance with 
the labeled recommendations for nivolumab in the European SmPC or the US PI [90, 
91]; see also Section 6.4.  
 In case of infusion-related reactions and hypersensitivity reactions, please refer to 
Section 12.4. 
 In case of immune-related adverse events related to EO2040 and/or nivolumab, 
instructions for treatment modifications as outlined in Section 6.4 should be followed.  
6.4 
Treatment modifications for EO2040 and nivolumab 
 At related Grade ≥3 adverse events special procedures are to be followed, see Section 
4.3. 
 Dose modifications for EO2040 include withholding of doses and treatment 
discontinuation (see Section 4.3, Section 5.3.3, and Section 6.4.1). Specifically for local 
administration site reactions there is usually no need for withholding doses; however, at 
any doubt the treating physician should contact the Medical Monitor for discussion and 
agreement regarding possible treatment delays, or modified injection site as compared 
to plan (see Section 6.3). 
 In instances where the protocol prescribes that EO2040 should be withheld or 
discontinued due to safety events, nivolumab might be continued, provided the treating 
physician considers this to be safe for the patient, and the continuation is approved by 
the Sponsor's medical monitor. 
 The only adjustments regarding dose for nivolumab are withholding of one or several 
doses, or discontinuation of treatment; such measures should be taken in accordance with 
the labeled recommendations for nivolumab in the European SmPC or the US PI [90, 
91]; see also Section 6.4.1 and Section 12.5.   


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 In instances where the label(s) for nivolumab [90, 91] prescribe that the drug should be 
withheld or discontinued due to safety events, EO2040 might be continued, provided the 
treating physician considers this to be safe for the patient, and the continuation is 
approved by the Sponsor's medical monitor. 
Dose adjustments are necessary to consider in the case of administration-related reactions 
(see Section 12.4). 
In case of immune-related adverse events related to EO2040 and/or nivolumab, instructions 
for treatment modifications as outlined in Section 6.4.1 should be followed.  
General criteria for treatment discontinuation and study participation termination are 
outlined in Section 5.3. 
6.4.1 
Recommended treatment modifications for EO2040 and nivolumab in 
case of immune-related adverse reactions   
For suspected immune-related adverse reactions, adequate evaluation should be performed 
to confirm etiology or exclude other causes. Based on the severity of the adverse reaction, 
the treatment should be withheld, and corticosteroids administered. If immunosuppression 
with corticosteroids is used to treat an adverse reaction, a taper of at least 1-month duration 
should be initiated upon improvement. Rapid tapering may lead to worsening or recurrence 
of the adverse reaction. Non-corticosteroid immunosuppressive therapy should be added if 
there is worsening or no improvement despite corticosteroid use. Table 7 shows the 
recommended treatment management for different immune-related adverse reactions. 
Regarding management of immune-related adverse reactions due to nivolumab see also the 
European SmPC or US PI [90, 91].  
Regarding nivolumab, patients should be monitored continuously (at least up to 5 months 
after the last dose) as an adverse reaction with nivolumab may occur at any time during or 
after discontinuation of therapy. There is no such timeline established for EO2040. However, 
it is assumed that the monitoring period for nivolumab would also be adequate for EO2040.  
Patients will be reviewed on a case-by-case basis, should they need to discontinue EO2040 
and/or nivolumab due to toxicity. See Section 6.4, regarding guidance for continuation of 
individual components of the used compounds in this trial. 
In case of detected, or suspicious, immune-related adverse reactions it is recommended that 
the treating physician consult at least one of the following guidance documents for a 
thorough description regarding possible workup for final diagnosis: 
 Management of toxicities from immunotherapy: ESMO Clinical Practice Guidelines 
for diagnosis, treatment and follow-up. Annals of Oncology 2017; 28 (Supplement 
4): i119–i142 [105] 
 Management of Immune-Related Adverse Events in Patients Treated With Immune 
Checkpoint Inhibitor Therapy: ASCO Guideline Update. J Clin Oncol 2021; 39 (36): 
4073-4126 [106] 
 NCCN Clinical Practice Guidelines in Oncology: Management of Immunotherapy-
Related 
toxicities. 
Version 
4.2021 
- 
September 
27, 
2021. 


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https://www.nccn.org/professionals/physician_gls/pdf/immunotherapy.pdf. 
Accessed on January 16, 2022 [107] 
The documents indicated above also includes specific guidance regarding specific treatment 
of diagnosed immune-related adverse reactions (e.g. substitution therapies with hormones, 
recommended local or systemic steroid doses etc. in context of the different and varied 
reactions possible) which is recommended as a basis for treatment decisions [105, 106, 107]. 
In addition, the European SmPC and the US PI also includes treatment recommendations for 
some immune-related immune reactions [90, 91], and Section 12.5 outlines management 
algorithms regarding nivolumab for some specific adverse events. 
Table 7 : Recommended treatment modifications for EO2040 and nivolumab in 
relation to immune-related adverse reactions 
Immune-related adverse 
reaction 
NCI-CTCAE v. 5.0 (123) 
Treatment action 
Immune-related pneumonitis 
Grade 2 
Withhold dose(s) until 
symptoms resolve, radiographic 
abnormalities improve, and 
management with 
corticosteroids or empirical 
antibiotics is complete. 
Grades 3 and 4 
Permanently discontinue 
treatment. 
Immune-related colitis 
Grade 2 diarrhea or colitis 
Withhold dose(s) until 
symptoms resolve and 
management with 
corticosteroids, if needed, is 
complete. 
 
Grade 3 diarrhea or colitis 
Withhold dose(s) until 
symptoms resolve and 
management with 
corticosteroids is complete. 
 
Grade 4 diarrhea or colitis 
Permanently discontinue 
treatment. 
Immune-related hepatitis 
AST or ALT > 3.0 to ≤ 5 × ULN, 
total bilirubin > 1.5 to ≤ 3.0 
×ULN 
Withhold dose(s) until 
laboratory values return to 
baseline and management with 
corticosteroids, if needed, is 
complete. 
 
AST or ALT > 5 × ULN. Total 
bilirubin >3 × ULN 
 
Permanently discontinue 
treatment. 
Immune-related nephritis and 
renal dysfunction 
Grade 2 (>1.5 - 3.0 x baseline; 
>1.5 - 3.0 x ULN) or Grade 3 
(>3.0 x baseline; >3.0 - 6.0 x 
ULN) creatinine elevation 
Withhold dose(s) until 
creatinine returns to baseline 
and management with 
corticosteroids is complete. 
Grade 4 (>6.0 x ULN) creatinine 
elevation 
Permanently discontinue 
treatment. 
Immune-related 
endocrinopathies 
 
Symptomatic Grade 2 or 3 
hypothyroidism, hyperthyroidism 
 
* Grade 1-3 
hypophysitis/hypopituitarism that 
Withhold dose(s) until 
symptoms resolve and 
management with 
corticosteroids (if needed for 
symptoms of acute 


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Immune-related adverse 
reaction 
NCI-CTCAE v. 5.0 (123) 
Treatment action 
is also associated with 
corresponding abnormal lab 
and/or pituitary scan 
 
Grade 2 adrenal insufficiency ^ 
 
* Grade 2 or 3 requiring initiation 
or change in daily management  
inflammation) is complete. 
Treatment should be continued 
in the presence of hormone 
replacement therapy as long as 
no symptoms are present. 
Grade 4 hypothyroidism 
Grade 4 hyperthyroidism 
* Grade 4 
hypophysitis/hypopituitarism 
* Grade 3 or 4 adrenal 
insufficiency or adrenal crisis ^ 
Grade 4 hyperglycemia 
Permanently discontinue 
treatment. 
Immune-related skin adverse 
reactions 
* Grade 2 rash covering > 30% 
body surface area 
Withhold dose(s) until rash 
reduces to ≤ 10% body surface 
area 
Grade 3 rash 
Withhold dose(s) until 
symptoms resolve and 
management with 
corticosteroids is complete. 
Grade 4 rash  
Permanently discontinue 
treatment. 
Stevens-Johnson syndrome or 
toxic epidermal necrolysis 
* or drug reaction with 
eosinophilia and systemic 
symptoms (DRESS) 
Permanently discontinue 
treatment. 
Immune-mediated encephalitis 
Monitor for changes in 
neurologic function 
 
Withhold for new-onset 
moderate to severe neurological 
signs or symptoms provided the 
symptoms are not related to the 
patients underlying CNS 
malignancy, or study treatment 
mechanism based immune 
attack on the tumor under 
treatment (in the latter cases 
standard of care for CNS 
malignancies should be 
implemented; see also Section 
6.8.2). Permanently discontinue 
study treatment for any grade 
drug related encephalitis. 
* Guillain-Barre Syndrome  
Any grade 
Permanently discontinue 
treatment. 
* Myasthenia Gravis 
Any grade 
Permanently discontinue 
treatment. 
* Other potentially immune-
mediated neurological diseases 
Myelitis, any grade 
Withhold dose(s), after workup 
if myelitis is not drug related, 
treatment may resume when AE 
resolves. 
Myelitis, any grade drug-related 
Permanently discontinue 
treatment. 


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Immune-related adverse 
reaction 
NCI-CTCAE v. 5.0 (123) 
Treatment action 
Other condition, grade 2 
Withhold dose(s), treatment 
may resume when AE resolves 
to baseline. 
Other condition, grade 3 or 4 
Permanently discontinue 
treatment. 
* Immune-related uveitis 
Grade 2 uveitis 
Withhold dose(s), treatment 
may resume if uveitis respond to 
topical therapy (eye drops) and 
uveitis resolves to Grade ≤ 1 or 
baseline. If patient requires oral 
steroids for uveitis, then 
permanently discontinue 
treatment. 
Grade 3 or 4 uveitis  
Permanently discontinue 
treatment. 
Immune-related myocarditis 
Grade 2 myocarditis;  
 
* symptoms induced from mild to 
moderate activity exertion 
 
 
Withhold dose(s) until 
symptoms resolve and 
management with 
corticosteroids is complete.  
 
Grade 3 or 4 myocarditis; 
 
* severe or life threatening, with 
symptoms at rest of with minimal 
activity or exertion, and/or where 
intervention indicated  
Permanently discontinue 
treatment 
Other immune-related adverse 
reactions 
Grade 3 (first occurrence) 
 
Withhold dose(s).  
 
 
Grade 4 or recurrent Grade 3; 
persistent Grade 2 or 3 despite 
treatment modification; inability 
to reduce corticosteroid dose to 
10 mg prednisone or equivalent 
per day 
Permanently discontinue 
treatment. 
 
Abbreviations: ALT = alanine aminotransferase; AST = aspartate aminotransferase, NCI-CTCAE = National Cancer 
Institute-Common Terminology Criteria for Adverse Events; ULN = upper limit of normal. 
Source: Opdivo SmPC [91], also stating toxicity grades are in accordance with NCI-CTCAE v4.0, and when text 
preceded by * updated based on standard protocol guidance from Bristol-Myers Squibb (market authorization holder for 
Opdivo). 
6.5 
Study treatment accountability, reconciliation, and return 
The Sponsor will be responsible for ensuring that the quality of the study treatment is 
adequate for the duration of the study. 
It is the responsibility of the Investigator or designee to ensure that the study treatment is 
only dispensed to the adequate patient. The study treatment must be dispensed from official 
study sites by authorized personnel according to local regulations. The Investigator or 
designee must maintain accurate records of the study treatment receipt, dispensing 
information, and disposition. 
Upon completion and termination of the study, all unused and/or partially used study 
treatment must be returned to the Sponsor or other authorized party. If return to Sponsor not 


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authorized by the Sponsor, study treatment shall be destroyed at the site following written 
confirmation by the Sponsor.  
If study treatment is to be destroyed at the site, it is the Investigator’s or designee’s 
responsibility to ensure that arrangements have been made for disposal, drug accountability 
has been completed by the site monitor, procedures for proper disposal have been established 
according to applicable regulations, guidelines, and institutional procedures, and appropriate 
records of the disposal have been documented and provided to the Sponsor or designee. 
All study treatments returned to the Sponsor or other authorized party must be accompanied 
by the appropriate documentation and be clearly identified. Study treatment may only be 
returned after drug accountability is completed. Returned supplies should be in their original 
containers (component vials that have clinical labels attached). 
Empty vials should not be returned to the Sponsor. Empty vials may not be destroyed until 
drug accountability is completed. It is the Investigator’s responsibility to arrange disposal of 
all empty vials according to the institutional regulations. 
The return or destruction of unused study treatment should be arranged by the site monitor. 
Further guidance and information for the final disposition of unused study treatment are 
provided in the Pharmacy Manual. 
6.6 
Method of treatment assignment  
This is an open-label (i.e. no blinding procedure will be implemented), non-randomized trial 
and each newly enrolled patient (upon signing the ICF) will be assigned a unique patient 
identification number (identifying trial, site, and patient), and assigned to the treatment after 
approval of eligibility by the Sponsor Medical Monitor according to the separate Cohort 
Administration Charter. 
Of special note: 
 If screening laboratory procedures were done within 72 hours prior to Visit 1, they 
do not need to be repeated at V1 unless it should be done per the Investigator’s 
opinion. If not done within 72 hours before V1, they need to be repeated at V1, and 
if there is a change of laboratory values from screening until V1 making the patient 
non-eligible the treating physician MUST not initiate treatment and inform the 
Enterome Medical Monitor immediately for decision on further actions.  
6.7 
Ancillary treatments 
6.7.1 
Prior and concomitant treatments and procedures 
Prohibited treatments prior to trial enrollment are outlined in the exclusion criteria (see 
Section 5.2). 
The Investigator must record the use of all prior and concomitant treatments and procedures, 
including medications and vaccines, taken during 28 days prior to signing informed consent. 
Anti-CRC treatments and procedures will be recorded as accurately as possible for the time 
from the diagnosis of the disease, i.e. for a longer time than 28 days prior to signing the 
informed consent. Likewise, for the duration of the study both prescribed and "over-the-
counter" medications, including herbal remedies, should be recorded in the source 
documents and eCRF along with: 


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 reasons for use, 
 dates of administration including start and end dates, and 
 dosage information, including dose, frequency, and route of administration. 
In case of surgical procedures, or other relevant non-drug medical interventions and 
procedures, all details must be collected in the eCRF in a similar manner as for medications 
per above. 
The Medical Monitor should be contacted if there are any questions regarding concomitant 
or prior therapy. 
Patients should be discouraged from starting any new medication, both prescribed and 
over-the-counter, including herbal remedies, without consulting the Investigator unless the 
new medication is required for emergency. 
6.7.2 
Permitted concomitant medications 
All non-cancer therapies that the Investigator feels appropriate are allowed in this study, 
except for the medications outlined in protocol Section 6.7.3. Thus, patients should receive 
full supportive care during participation in the trial and standard of care treatment per local 
practice and according to the judgment of the Investigator or treating physician. 
Administration of steroids through a route known to result in a minimal systemic exposure 
(topical, intranasal, intro-ocular, or inhalation) is acceptable. 
Of note, the use of systemic corticosteroids and other immunosuppressants (beside trial 
treatment) should be avoided because of their potential interference with the 
pharmacodynamic activity of the investigational treatments. However, the use of systemic 
corticosteroids and other immunosuppressants might be of vital importance for patient safety 
in relation to treatment of adverse reactions (see Section 6.4 and Section 12.5). Also, steroid 
medication in relation to administration-related reactions might be vital (see Section 12.4). 
The following is general guidance regarding steroid treatment: 
 If possible, restrict use of systemic steroids to a dexamethasone dose < 2 mg/day or 
equivalent (i.e. 13 mg/day of prednisone, or 53 mg/day of hydrocortisone), unless 
required to treat an adverse event (see above).  
 Adrenal replacement steroid doses > 2 mg/day dexamethasone (i.e. 53 mg/day of 
hydrocortisone) is permitted in patients needing steroid replacement therapy but 
should be discussed with the Sponsor. 
In any case of steroid administration, it is of special importance to record dosage, frequency, 
dates of administration, and route of administration in the eCRF.  
 
Recommendations regarding vaccination against SARS-CoV-2/COVID-19 
 The Sponsor recommends strongly that patients if possible are vaccinated against SARS-
CoV-2/COVID-19 before inclusion into the study. If vaccine is available for only one 
vaccine administration before study inclusion this is also recommended, and in such 
cases the timing of the second vaccine administration (and potential further doses) are to 
be discussed and agreed with the Medical Monitor. The recommended time interval 
between a COVID-19 vaccine administration and the initial study drug administration is 
2 weeks, however, other trial exclusion criteria should also be considered (e.g. regarding 


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patients with persistent toxicities) when assessing possible timing of start of study 
treatment in relation to COVID-19 vaccine administration. 
 For patients who have not been vaccinated against SARS-CoV-2/COVID-19 before 
study inclusion, but vaccine becomes available for them during their study participation, 
or a booster dose of SARS-CoV-2/COVID-19 vaccine is deemed necessary, the Sponsor 
also strongly recommends vaccination. For such patients the Sponsor recommends 
administration of the first dose of the COVID-19 vaccine during the boosting phase 
(EO2040 administered every 4 weeks), 2 weeks after the latest EO2040 administration 
(and thereby 2 weeks before the next EO2040 administration), and in a different location 
[Note, if this time schedule would lead to unnecessarily long waiting time for COVID-
19 vaccination, alternative administration time points should be discussed with the 
Medical Monitor]. 
 The currently (2021-03-24) by US Food and Drug Administration (FDA) and European 
Medicines Agency (EMA) approved COVID-19 vaccines are acceptable for use in the 
trial (local restrictions per country specific authority of course takes precedence): 
 
COVID-19 Vaccine 
developer/ 
manufacturer 
Vaccine 
platform 
Type of 
candidate 
vaccine 
Number 
of doses 
(base) 
Timing 
between 
doses* 
Route of 
administration 
University of 
Oxford/AstraZeneca 
Non- 
Replicating 
Viral Vector 
ChAdOx1-S 
2 
28 days 
IM 
Moderna/NIAID 
RNA 
LNP- 
encapsulated 
mRNA 
2 
28 days 
IM 
BioNTech/Fosun 
Pharma/Pfizer 
RNA 
3 LNP-mRNAs 
2 
21 days 
IM 
Johnson & Johnson/ 
Janssen 
Non- 
Replicating 
Viral Vector 
Ad26.COV2-S 
1 
NA 
IM 
* Local country/site timing between doses might be different from what is stated in the table. 
6.7.3 
Prohibited medications and other therapies 
The use of systemic corticosteroids and other immunosuppressants should be avoided 
because of their potential interference with the pharmacodynamics activity of the study 
treatment. However, in some cases, systemic corticosteroids cannot be avoided; guidance 
regarding steroid use for patients participating in the trial is therefore outlined in Section 
6.7.2. 
The use of live or attenuated vaccine therapy for prevention of infectious diseases during 
treatment with study medication and for at least 3 months after end of treatment with study 
medication should be avoided. If there is no alternative, to live or attenuated, vaccine 
available, and it is considered of utmost importance for the individual patient to receive the 


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vaccine, an individual benefit/risk assessment should be made regarding the duration of 
"wash-out" period necessary after termination of study treatment, and study treatment should 
be stopped as applicable.   
Patients should be discouraged from taking medications, including herbal remedies, with 
immunostimulatory properties, or known to potentially interfere with major organ function, 
during trial participation. If needed, any such possible therapy should be discussed between 
the treating physician and the Medical Monitor of the Sponsor. 
During trial participation patients should not receive any other anti-CRC treatment than the 
specified trial treatments, and treatments outlined in Section 6.7.2.  
If any other anti-CRC treatment, or procedure, would be considered for start in an individual 
patient, it is the duty of the Investigator to discuss the consideration with the Medical 
Monitor of the Sponsor for a decision on how to handle continued trial specific treatment 
and trial participation.  
Start of non-trial systemic anti-CRC treatments would lead to censoring of the patient for 
relapse related endpoints at the time of start of the therapy; or assessment of the patient as 
having relapsed disease if relevant criteria would be fulfilled.  
6.7.4 
Contraception 
Potential postmenopausal status for female patients will be confirmed with a screening 
serum follicle-stimulating hormone (FSH) level > 40 mIU/mL. Rules for contraception 
during study participation are outlined in the inclusion criteria, see Section 5.1. 
6.8 
Treatment compliance 
Since all study related treatments are administered at the clinical site, the Investigator or 
designee must maintain accurate records of all study treatments, including as applicable 
dates of study drug receipt, quantities received and dispensed, and batch/lot numbers. In 
addition, study related treatments must be noted in the patient’s medical records and eCRF, 
with the date and time of administration and dose of each study treatment, alternatively 
information regarding dispense documented. Based on documentation per above treatment 
compliance is going to be monitored and reported.  
 
 
 
 


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7 
STUDY ASSESSMENTS AND PROCEDURES 
7.1 
Study schedule 
Study assessments and procedures, and their timings (including visit windows) are 
summarized in the Study Schedule (Table 8). As protocol exemptions are not foreseen in 
this trial, except in case of immediate safety concerns, any deviation from the planned study 
schedule should be discussed with the Sponsor immediately upon occurrence or awareness 
to determine if the patient should continue, or discontinue, the study treatment. Adherence 
to the study design requirements, including those specified in the Study Schedule, is essential 
and required for adequate study conduct. 
Note, for safety reasons, unscheduled visits, and unscheduled investigations, in the best 
interest of the patient per judgement of the treating physician might be instituted at any time 
during trial participation for an individual patient. 
 
 
 


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Table 8 : Study Schedule 
EOCRC1-22 STUDY SCHEDULE 
1 
 
Screen 
Treatment period 
(visit window +/-2 days if nothing else stated for the specific item; see footnotes) 
Safety 
visit 
Safety 
visit 
Post-treatment 
follow-up 
Visit number 
 
S 
1 
S 
2 
V 
1  
V 
2 
V 
3 
V 
4 
no 
visit 
V 
5 
V  
6 
V 
7 
no 
visit 
V 
8 
no 
visit 
V 
9 
no 
visit 
V 
10 
V   
d30 
V  
d100 
before  
PD 32 
after  
PD 30, 32 
Study day 
 
(*D -28 to -1) 
 
- 
 
* 
D 
1  
D 
15  
D 
29  
D 
43 
D 
57 
D 
71 
D  
85 
D  
99 
D  
113 
D 
127 
D 
141 
D  
155 
D  
169 
D 
183 
30 days 
(-0/+4) 
after last 
trial trt 
100 days  
(-0/+7) 
after last 
nivolumab 
every 3 
months +/- 7 
days 
every 3 
months +/- 7 
days 
Study week                      
(green color = week of CT & ctDNA) 
- 
- 
W1 
W3 
W5 
W7 
W9 
W11 
W13 
W15 
W17 
W19 
W21 
W 23 
W25 
W27 
- 
- 
- 
- 
Informed Consent Form #1 2  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion criteria 2 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent Forms #2 3 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 3  
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TREATMENTS 
Cohort 1: EO2040 
 
 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
 
Cohort 1: nivolumab/dose in mg 
 
 
X/240 X/240 X/240 X/480 
 
X/480 
 
X/480 
 
X/480 
 
X/480 
 
 
 
 
 
 
Cohort 2A: EO2040 
 
 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
 
Cohort 2A: nivolumab/dose in mg 4 
 
 
 
 
 
 
 
 
 
X/480 4 
 
X/480 4 
 
X/480 4 
 
 
 
 
 
 
 
EXAMINATIONS 
Age and abbreviated medical history 
2 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HLA-testing 2, 5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Complete medical history 6 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prior medications/treatments 7 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant medication/treatment 8 
 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
X 31 
X 31 
Height 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight, ECOG performance status 
 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
 
 
Vital signs 9 
 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
 
 
Physical examination 10 
 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
 
 
Safety assessment; AEs/SAEs 11, 12 
X 
X 
Continuous during study treatment and until the Vd30 and Vd100, respectively (see Section 8.3.1) 
X 
X 
X 12 
X 12 
Hematology assessment 13, 14 
 
X 
X15 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
 
 
Coagulation assessment 13, 16 
 
X 
X15 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
 
 
Serum chemistry assessment 13, 17 
 
X 
X15 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
 
 
Urinalysis assessment 13, 18 
 
X 
X15 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
 
 


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EOCRC1-22 STUDY SCHEDULE 
1 
 
Screen 
Treatment period 
(visit window +/-2 days if nothing else stated for the specific item; see footnotes) 
Safety 
visit 
Safety 
visit 
Post-treatment 
follow-up 
Visit number 
 
S 
1 
S 
2 
V 
1  
V 
2 
V 
3 
V 
4 
no 
visit 
V 
5 
V  
6 
V 
7 
no 
visit 
V 
8 
no 
visit 
V 
9 
no 
visit 
V 
10 
V   
d30 
V  
d100 
before  
PD 32 
after  
PD 30, 32 
Study day 
 
(*D -28 to -1) 
 
- 
 
* 
D 
1  
D 
15  
D 
29  
D 
43 
D 
57 
D 
71 
D  
85 
D  
99 
D  
113 
D 
127 
D 
141 
D  
155 
D  
169 
D 
183 
30 days 
(-0/+4) 
after last 
trial trt 
100 days  
(-0/+7) 
after last 
nivolumab 
every 3 
months +/- 7 
days 
every 3 
months +/- 7 
days 
Study week                      
(green color = week of CT & ctDNA) 
- 
- 
W1 
W3 
W5 
W7 
W9 
W11 
W13 
W15 
W17 
W19 
W21 
W 23 
W25 
W27 
- 
- 
- 
- 
TSH, and free T4 13, 19 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
 
 
S-Cortisol 13, 19 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
 
 
FSH test; when applicable 13, 20 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test; as applicable 13, 21 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X22 
X22 
X22 
X22 
HIV, hepatitis B, and hepatitis C 
status 13, 23 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ECG 24 
 
X 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X 
 
 
 
PROCEDURES FOR IMMUNOLOGICAL ASSESSMENTS, TISSUE COLLECTION, AND EXPLORATORY ENDPOINTS 
Stools sample; delivery of kit for 
sample collection 25 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Stools sample; collection of sample 
collection 25 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Collecting tumor tissue  for setup of 
Signatera assay 37 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sampling whole blood for setup of 
Signatera assay 37 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Collection of formalin-fixed, 
paraffin-embedded tumor tissue 26 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tumor tissue collections 27 
 
 
Tumor tissue collection at any time during study treatment when medically indicated procedure and possible 
 
 
 
 
PBMC/serum collection/preparation 
28 
 
X 
X 
X 
X 
X 
 
X 
X 
 
 
X 
 
 
 
X 
 
 
X 28 
 
Plasma collection/preparation 36 
 
 
X 
X 
X 
 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
X36 
 
 
TUMOR EVALUATIONS AND SURVIVAL ASSESSMENTS 
Serum  CEA 35 
 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
X 
 
 
X35 
 
ctDNA  with the Signatera assay, 
plasma collection/preparation 33 
 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
X 
 
 
X 33 
 
CT chest, abdomen, pelvis 29, 34 
 
X 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
X 29 
 
Survival status 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 


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Abbreviations: APTT = activated partial thromboplastin time: AE = adverse event; ALP = alkaline phosphatase; ALT = alanine aminotransferase; anti-HBcAg = antibody to the hepatitis B core 
antigen; anti-HBsAg = antibody to the hepatitis B surface antigen; AST = aspartate aminotransferase; BP = blood pressure; bpm = beats per minute; BUN = blood urea nitrogen; CRP = C-reactive 
protein;; ECG = electrocardiogram; FSH = follicle-stimulating hormone; Hb = hemoglobin; HBsAg = hepatitis B surface antigen; HIV = human immunodeficiency virus; HLA = human leukocyte 
antigen; HR = heart rate; INR = international normalized ratio; NCI-CTCAE = National Cancer Institute-Common Terminology Criteria for Adverse Events; PBMC = peripheral blood mononuclear 
cells; PCR = polymerase chain reaction; PD = progressive disease; PS = performance status; RBC = red blood cells; S = screening; SAE = serious adverse event; T = thyroxine; TSH = thyroid-
stimulating hormone; V = visit; W = week; WBC = white blood cells. 
1. 
If additional investigations (specified in the study schedule but repeat, or unplanned, or not specified in the study schedule) are performed in the context of normal care for the patients included in 
the trial and such investigations are important for the understanding and evaluation of the impact of the trial treatment and outcome (e.g. regarding safety and/or efficacy) such information will be 
collected within the scope of the trial. 
2. 
See Section 4.2 regarding the applied 2-stage consent procedure for the trial; the initial minimized consent is mainly related to the procedure of HLA-testing, and before testing also establishing, 
based on available information, that the patient has eligible CRC, and an age ≥ 18 years (inclusion criteria #1-7; see Section 5.1). HLA-testing can be performed within the scope of the trial, but 
HLA-information based on testing before the screening period is also acceptable. Abbreviated medical history means collection of history of CRC, including treatment history, at a detailed enough 
level to enable assessment of inclusion criteria #2-5 (see Section 5.1). 
3. 
The second stage of the consent procedure will be related to trial treatment and follow-up; for remaining inclusion/exclusion criteria see Section 5.1 and Section 5.2. 
4. 
Nivolumab to be added ONLY if ctDNA clearance is NOT ACHIEVED at week 13. 
5. 
HLA-testing will be performed locally (see also Section 7.2). Information regarding the local lab, including methods used will be collected. All information available regarding the individual 
patient MHC-composition will be collected, i.e. also information beside the HLA-A type. Patients who are not HLA-A2 positive will have “screen failure”-status.  
6. 
Includes demographics (including race and ethnicity), extended history of hematology/oncology disease(s) (see Section 7.3), including treatment history, and history of other disease processes 
(active or resolved), including concomitant illness. 
7. 
Medications, including vaccines, and other relevant treatments (e.g. surgeries) during 28 days prior to signing informed consent will be collected. All applicable treatments (systemic and local), 
also before 28 days prior to informed consent signing, for hematology/oncology disease(s) will be collected. 
8. 
All ongoing medications and treatments will be collected. 
9. 
Vital signs include temperature, BP, and HR. BP and HR will be measured according to local standard practice. If systolic BP is below 100 mmHg or above 150 mmHg, and/or diastolic BP is 
below 50 mmHg or above 90 mmHg, the measurement will be repeated. The HR measurement will be repeated when below 50 bpm or above 100 bpm. 
10. Physical examination will include an examination of major body systems; general appearance, skin, neck (incl. thyroid), ENT (ears, nose, throat), lungs, heart, abdomen, lymph nodes (all major 
stations palpable), extremities, neurology, and other sites as applicable (i.e. symptom directed).  
11. Adverse events will be reported according to NCI-CTCAE v5.0. Documentation of AEs/SAEs starts after trial consent has been given by the patient. In case of death, an as precise as possible 
cause of death is going to be documented.  
12. After the Vd100 post-treatment visit for safety assessment (100 -0/+7 days after the last dose of nivolumab/placebo) has been made, only SAEs with plausible causal relationship with trial treatment 
will be documented. In addition, in case of death, an as precise cause of death as possible is going to be documented. 
13. Safety blood samples for hematology, coagulation, serum chemistry, urinalysis, hormones, and virus serology to be performed and evaluated locally at each timepoint indicated; samples to be 
drawn before administration of study treatments if scheduled for the same day.  
14. Hematology: Hb, hematocrit, platelet count, RBC count, WBC count, and differential (percentage and total neutrophil [including bands], lymphocyte, monocyte, eosinophil, and basophil counts); 
when indicated other blood cells (e.g. reticulocytes, etc.), might also be analyzed. 
15. If screening laboratory procedures were done within 72 hours prior to V1, they don’t need to be repeated unless necessary in the opinion of the Investigator. 
16. Coagulation: prothrombin time, INR, and PTT (or aPTT). 
17. Serum chemistry: albumin, total protein, calcium, sodium, potassium, chloride, glucose, AST, ALT, ALP, bilirubin, lactate dehydrogenase, creatinine, BUN (or serum urea), CRP; when indicated 
other serum parameters (e.g. conjugated and unconjugated bilirubin, gamma-glutamyl transpeptidase, uric acid, etc.), might also be analyzed. 
18. Urinalysis (urine dipstick, locally or centrally provided): protein, glucose, ketones, bilirubin, blood, and leucocytes; when indicated further assessments (e.g. microscopic evaluations of WBC, 
RBC, etc.) might also be done. 
19. Adrenal, pituitary, and thyroid gland function testing includes TSH, free T4, and serum-cortisol. If pathological outcome of TSH/free T4 testing further assessment including e.g. T3 should be 
done. If pathological outcome of serum-cortisol testing (as defined at the local institution), an ACTH stimulation test should be performed. Availability of laboratory results for the basic 


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adrenal/pituitary/thyroid function tests is not mandatory before study drug administration if repeat sampling after the initial study drug administration has been done. Further testing during, or after 
study treatment discontinuation as clinically indicated per judgment of the treating physician. 
20. For postmenopausal women: FSH measurement for confirmation of postmenopausal status.  
21. For women of childbearing potential: at screening highly sensitive serum pregnancy test within 72 hours before study treatment start, and thereafter tests to be done per the schedule. 
22. For female patients of childbearing potential, a serum pregnancy test will be conducted monthly during active study treatment, and at the first follow-up visit after permanent discontinuation of 
study treatment (30 days after completion of the last dose of study treatment). In addition, the pregnancy tests will continue to be done on a monthly basis until 6 months after study treatments 
have been stopped, if not a new anti-tumor treatment has been initiated earlier in which case the study related testing will be terminated at the time of start of new anti-tumor treatment.  
23. Assessment of HIV-status, hepatitis B (HBsAg, anti-HBcAg, anti-HBsAg), and hepatitis C (hepatitis C antibody). A polymerase chain reaction (PCR) viral detection test might also be used to 
confirm HCV status if deemed adequate by the treating physician in a patient with immunodeficiency (possible association with a false negative HCV antibody test result); in addition, all patients 
with a positive hepatitis C antibody test should be tested with a PCR test to prove if the patient is still to be considered infected (positive PCR test), or can be included in the trial (negative PCR 
test). 
24. A 12-lead ECG should be recorded according to local site standard procedures (+/- 4 days of the scheduled time). For all patients in the trial ECGs should be recorded as clinically indicated. If 
ECG and study treatment scheduled for the same day, the ECG should be performed before study treatment administration. 
25. A study kit to collect stools will be given to the patient at screening, and if the patient is eligible for the study, he/she will be asked to collect a stool sample any time prior to V1 (W1). If the patient 
fails screening, he/she will be asked to return the study kit to the site. A stool collection kit will also be provided to the patient at V4 (W7), and he/she will be asked to collect a stool sample any 
time prior to V5 (W11). 
26. Tissue samples to be collected if the individual patient has consented to this procedure; 30 unstained formalin-fixed, paraffin-embedded tissue sections, a 5-8 µm thickness, from the patient's tumor 
obtained at diagnosis or later during the disease (the newest available tissue sample would be preferred). The samples will be sent to a central laboratory for analyses as outlined in the 
objectives/endpoints of the trial (see Section 3). 
27. Tumor tissue to be collected at any time during study treatment when medically indicated procedure is made and it is possible to sample tissue, if the individual patient has consented to this 
procedure. The samples will be sent to a central laboratory for analyses as outlined in the objectives/endpoints of the trial (see Section 3). 
28. PBMC and serum will be sampled at the timepoints indicated (+/- 4 days), and before study treatment administration if scheduled on the same days. At each sampling time the target is to collect 
48 mL of blood for PBMC preparation, and 5 mL of blood for serum preparation. After week 27 continue PBMC/serum collections every 3 months (+/- 7 days) until ctDNA test showing positive 
result, or scan documented relapse (or until other anti-cancer treatment would be started before confirmed progressive disease), or for a maximum of 3 years from first study treatment. 
Preparation/shipping procedures for the blood and serum samples will be detailed in the Laboratory Manual; a central laboratory will handle the immune response analyses.  
29. Computer tomography (with contrast) of chest, abdomen, and pelvis will be performed at screening (if not done within scope of normal care; Note, if the possible SOC scanning is not done within 
-7 days/+28 days of the positive Signatera assay which is accepted as confirmatory test for the trial, the scanning needs to be repeated within the screening part #2 procedures), Week 13 (+/- 
4 days), and Week 27 (+/- 4 days). If local SOC includes MRI instead of CT, this is also acceptable, but scanning type should, if at all possible, be consistent for an individual patient. After week 
27, scanning is to be done every 3-6 months per standard of care for the patient's stage of CRC disease. Collection of information from a protocol perspective should be done until scan documented 
relapse of disease (or until other anti-cancer treatment would be started before confirmed progressive disease), or for a maximum of 3 years from first study treatment in the individual patient. See 
Section 1.1 regarding recommendations for surveillance after curative treatments.  
30. If the patient is unable to attend a site visit, follow-up via a telephone contact is acceptable. 
31. Collection of information regarding further anti-cancer treatments. 
32. Follow-up will at the longest continue in an individual patient until 3 years from first study treatment.  
33. Inclusion criteria demands presence of minimal residual disease as defined by positive ctDNA assay after completion of planned standard of care perioperative and/or adjuvant therapies. Patients 
may be identified for enrollment with any CLIA-certified, or similar certification outside of the USA, ctDNA assay for MRD. Note, MRD status will be confirmed with the Signatera assay prior 
to initiation of study therapy (i.e. within the screening part #2 procedures) for all patients who have not had a Signatera research grade testing where all parameters from the testing can be 
provided for the current trial. The ctDNA positive outcome test date should be more than 4 weeks after the finalization of all curative treatments, including chemotherapy. ctDNA testing 
within the study is done at Week 7 (+/- 4 days), Week 13 (+/- 4 days), and Week 27 (+/- 4 days). Thereafter, continue ctDNA testing every 3 months per local standard of care. Collection of 
information from a protocol perspective should be done until scan documented relapse of disease (or until other anti-cancer treatment would be started before confirmed progressive disease), or 
for a maximum of 3 years from first study treatment. 


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34. Note, at suspicion of relapse of CRC adequate investigations per standard medical practice should be performed to verify, or not, the suspicion; any used method (e.g. CT, MRI, endoscopy, etc.) 
and outcome should be documented in the eCRF throughout the trial. 
35. Serum carcinoembryonic antigen (CEA) to be tested (local lab) at the timepoints indicated (+/- 4 days). After week 27 continue CEA testing every 3 months (+/- 7 days) until ctDNA test showing 
positive result, or scan documented relapse (or until other anti-cancer treatment would be started before confirmed progressive disease), or for a maximum of 3 years from first study treatment. 
Note, if an individual patient earlier during treatment of his/her disease has shown a positive result for another serum measurable tumor marker than CEA, this tumor marker should be 
followed in addition to CEA. 
36. Plasma will be sampled at the timepoints indicated (+/- 4 days), and before study treatment administration if scheduled on the same days. At each sampling time the target is to collect 10 mL of 
blood for plasma preparation. After week 27 continue plasma collections every 3 months (+/- 7 days) until ctDNA test showing positive result, or scan documented relapse (or until other anti-
cancer treatment would be started before confirmed progressive disease), or for a maximum of 3 years from first study treatment. Preparation/shipping procedures for the samples will be detailed 
in the Laboratory Manual; plasma is mainly collected for possible characterization of changes in ctDNA allele fractions over time with therapy, but can also be used for assays related to other 
exploratory endpoints.  
37. For all patients who have not had a Signatera research grade testing where all parameters from the testing can be provided for the current trial. The specifics of samples and sampling procedures 
are outlined in the Laboratory Manual and not in this Study Schedule. 
 
 
 
 
 


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7.2 
Screening period #1 
The trial will include a 2-stage consent and screening procedure (see Section 4.2).  
The initial minimized consent is mainly related to the procedure of HLA-testing, and before 
testing also establishing, based on available information, that the patient has eligible CRC, 
and an age ≥ 18 years (inclusion criteria #1-7; see Section 5.1). Abbreviated medical history 
means collection of history of CRC, including treatment history, at a detailed enough level 
to enable assessment of inclusion criteria #2-7 (see Section 5.1). 
The following will be done during screening period #1: 
 
study ICF procedure for consent #1, and  
 
eligibility ascertained for inclusion criteria #1 to #7; for the disease this means to 
ascertain that the patient has eligible CRC, and an age ≥ 18 years, plus HLA-
testing. 
HLA-testing can be performed within the scope of the trial, but HLA-information based on 
testing before the screening period is also acceptable (no time limit is applied; patients with 
prior allogeneic hematopoietic stem cell transplantation are not eligible for the trial). 
HLA-testing will be performed locally; information regarding the local lab, including 
methods used will be collected.  
Low resolution HLA typing, i.e. typing of the HLA A loci 2 to 2 digits is sufficient for trial 
screening purposes, but 4 digits resolution (high resolution) typing of the HLA A loci 2 
should be conducted, if at all possible (but is not completely mandatory – see further below), 
on all patients who participate in the trial.  
If high resolution testing is slower (to get a response) than low resolution testing, a possibility 
is thus to first make a test with low resolution for trial screening, and then at a later point in 
time (or on the same sample if possible) also make a high-resolution testing. There is a local 
site judgment, in relation to other screening procedures, to be made regarding what is “too 
slow” in relation to utilizing low- or high-resolution testing; but as a general guidance it is 
assumed that a result for HLA testing for screening purposes should be available within 
approximately a week. If for local reasons it is not possible to manage high resolution testing 
(e.g. due to sampling restrictions, testing abilities, or anything else), either for screening, or 
at a later point in time (per above), it is acceptable with low resolution testing only.  
All information available regarding the individual patient MHC-composition will be 
collected, i.e. also information beside the HLA-A type. Patients who are not HLA-A2 
positive will be assigned a “screen failure”-status and not continue any further trial 
procedures; such patients will be replaced. 
7.3 
Screening period #2 
The second stage of the consent procedure, and screening period #2, will be related to trial 
treatment and follow-up aspects of the trial procedures, except those outlined in Section 7.2 
(for remaining inclusion/exclusion criteria see Section 5). All timelines included in the 
protocol and related to the signature of the ICF are referring to the signature of this second 
part of the ICF. All evaluations must be completed and reviewed within the timeframe 
defined in the Study Schedule (Table 8) to confirm that potential patients meet all eligibility 
criteria. The Investigator will record adequate details of all patients screened and to confirm 
eligibility, or record reasons for screen failure, as applicable (see Section 5.3.2). 


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Procedures conducted as part of the patient’s routine clinical management (e.g. blood counts) 
and obtained before signing the consent form may be utilized for screening purposes, 
provided the procedure met the protocol-specified criteria. Results obtained also the week 
before screening period #2 started (i.e. from study day -35 per the Study Schedule) can be 
used for study purposes provided approved by the study Medical Monitor.  
All screening examinations will be performed locally, at the site or an affiliate site due to 
e.g. the location of the site and the patient.  
The maximum amount of blood to be taken from each patient at screening, or over the 
duration of the study, including any extra assessments that may be required, will not exceed 
volumes as stated by local regulations. Repeat, or unscheduled samples, may be taken for 
safety reasons, or for technical issues with the samples. 
Patients who do not fulfill all eligibility criteria will be assigned a “screen failure”-status and 
not continue any further trial procedures; such patients will be replaced.  
The following will be done during screening period #2: 
 
study ICF procedure for consent #2,  
 
eligibility ascertained for inclusion and exclusion criteria (see Section 5), 
 
documentation of  
o patient demographics (including race and ethnicity), 
o extended history of hematology/oncology disease(s), including dates of 
diagnosis and treatments history, and especially for all relevant 
information for adequate grade, stage, and prognostic system evaluations 
of the CRC, 
o medical history of other (than hematology/oncology) disease processes 
(all prior significant illnesses that are relevant to patient safety or that the 
patient has experienced prior to screening; especially history of 
anaphylaxis or other reactions following vaccination, and history of 
immune deficiencies), and concomitant illness (illnesses present at the 
time of informed consent are to be regarded as concomitant illnesses; 
illnesses first occurring or detected during the study and/or worsening of 
a concomitant illness during the study are to be documented as AEs on 
the eCRF and as SAEs if case seriousness criteria are met),  
o medications, including vaccines, and other relevant treatments (e.g. 
surgeries) during 28 days prior to signing informed consent; all applicable 
treatments (systemic and local), also before 28 days prior to informed 
consent signing, for hematology/oncology disease(s) will be collected,  
o concomitant medications/treatments,  
 
measurement/assessment of 
o height and weight, 
o ECOG performance status (see Section 12.1), 
o vital signs including temperature, blood pressure (BP), and heart rate 
(HR) (BP and HR will be measured according to local standard practice); 
if systolic BP is below 100 mmHg or above 150 mmHg, and/or diastolic 
BP is below 50 mmHg or above 90 mmHg, the measurement will be 
repeated; the HR measurement will be repeated when below 50 bpm or 
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o major body systems via physical examination; general appearance, skin, 
neck (incl. thyroid), ENT (ears, nose, throat), lungs, heart, abdomen, 
lymph nodes (all major stations palpable), spleen, extremities, neurology, 
and other sites as applicable (i.e. symptom directed),  
 
safety blood samples for hematology, coagulation, serum chemistry, urinalysis, 
hormones, and virus serology to be performed and evaluated locally, detailed as 
o hematology including Hb, hematocrit, platelet count, RBC count, WBC 
count, and differential (percentage and total neutrophil [including bands], 
lymphocyte, monocyte, eosinophil, and basophil counts); when indicated 
other blood cells (e.g. reticulocytes, etc.), might also be analyzed, 
o coagulation including prothrombin time, international normalized ratio 
(INR), and partial thromboplastin time (PTT) (or activated PTT; aPTT), 
o serum chemistry including albumin, total protein, calcium, sodium, 
potassium, chloride, glucose, AST, ALT, ALP, bilirubin, LDH, 
creatinine, BUN (or serum urea), CRP; when indicated other serum 
parameters (e.g. conjugated and unconjugated bilirubin, gamma-glutamyl 
transpeptidase, uric acid, total protein, etc.), might also be analyzed, 
o urinalysis (urine dipstick) including protein, glucose, ketones, bilirubin, 
blood, and leucocytes; when indicated further assessments (e.g. 
microscopic evaluations of WBC, RBC, etc.) might also be done, 
o assessment of TSH, and free T4 for evaluation of thyroid function, if 
pathological outcome of TSH/free T4 testing further assessment 
including e.g. T3 should be done, 
o assessment of serum cortisol for evaluation of potential adrenal 
insufficiency; if pathological outcome of serum-cortisol testing (as 
defined at the local institution), an ACTH stimulation test should be 
performed,  
o assessment of HIV-status, hepatitis B (HBsAg, anti-HBcAg, anti-
HBsAg), and hepatitis C (hepatitis C antibody); a polymerase chain 
reaction (PCR) viral detection test might also be used to confirm HCV 
status if the patient by the treating physician is assessed as having an 
immunodeficiency as this condition can be associated with a false 
negative HCV antibody test result; in addition, all patients with a positive 
hepatitis C antibody test should be tested with a PCR test to prove if the 
patient is still to be considered infected (positive PCR test), or can be 
included in the trial (negative PCR test), 
o for women of childbearing potential, highly sensitive serum pregnancy 
test,  
o for postmenopausal women, FSH measurement for confirmation of 
postmenopausal status, 
 
a study kit to collect stools will be given to the patient at screening, and if the 
patient is eligible for the study, he/she will be asked to collect a stool sample any 
time prior to V1 (W1) (if the patient fails screening, he/she will be asked to return 
the study kit to the site), 
 
if the individual patient has consented to the procedure, tissue samples will be 
collected; a maximum of 30 unstained formalin-fixed, paraffin-embedded tissue 
sections, a 5-8 µm thickness, from the patient’s tumor obtained at diagnosis or 


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later during the disease (the newest available tissue sample would be preferred) - 
the samples will be sent to a central laboratory for analyses, 
 
PBMC and serum will be sampled; at each sampling time the target is to collect 
48 mL of blood for PBMC preparation, and 5 mL of blood for serum preparation 
- preparation/shipping procedures for the blood and serum samples will be 
detailed in the Laboratory Manual; a central laboratory will handle the immune 
response analyses,  
 
plasma will be sampled; at each sampling time the target is to collect 10 mL of 
blood for plasma preparation - preparation/shipping procedures for the samples 
will be detailed in the Laboratory Manual,  
 
serum carcinoembryonic antigen (CEA) to be tested (local lab); note, if an 
individual patient earlier during treatment of his/her disease has shown a 
positive result for another serum measurable tumor marker than CEA, this 
tumor marker should be followed in addition to CEA, 
 
inclusion criteria demands presence of minimal residual disease as defined by 
positive ctDNA assay; patients may be identified for enrollment with any CLIA-
certified, or similar certification outside of the USA, ctDNA assay for MRD; 
note, MRD status will be confirmed with the Signatera assay prior to initiation 
of study therapy (i.e. within the screening part #2 procedures) for all patients 
who have not had a Signatera research grade testing where all parameters from 
the testing can be provided for the current trial; the ctDNA positive outcome test 
date should be more than 4 weeks after the finalization of all curative treatments, 
including chemotherapy. 
 
a computer tomography (with contrast) of chest, abdomen, and pelvis will be 
performed at screening (if not done within scope of normal care; note, if the 
possible SOC scanning is not done within -7 days/+28 days of the positive 
Signatera assay which is accepted as confirmatory test for the trial, the scanning 
needs to be repeated within the screening part #2 procedures); if local SOC 
includes MRI instead of CT, this is also acceptable, but scanning type should, if 
at all possible, be consistent for an individual patient,  
 
a 12-lead electrocardiogram (ECG) will be recorded according to local site 
standard procedures, and 
 
adverse events will be reported according to NCI-CTCAE v5.0; documentation 
of AEs/SAEs starts after initial trial consent has been given by the patient.  
7.4 
Visits during study treatment 
Visits V1 until V10 are planned to be performed as outlined in Table 8, including visit 
windows of +/- 2 days if nothing else stated in Table 8 regarding specific items. 
Study treatments are to be administered as outlined in Section 4.3, Section 6.3, and Section 
6.4, and Table 8.  
Measurements/assessments/sampling as outlined below are to be done before administration 
of study treatments if scheduled for the same day, if no other specific order of events is 
defined.  
At all planned visits for treatments during the treatment period (see Table 8), the following 
items should be assessed and documented: 


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 
concomitant medications/treatments,  
 
weight, and ECOG performance status (see Section 12.1), 
 
vital signs including temperature, blood pressure (BP), and heart rate (HR) (BP 
and HR will be measured according to local standard practice); if systolic BP is 
below 100 mmHg or above 150 mmHg, and/or diastolic BP is below 50 mmHg 
or above 90 mmHg, the measurement will be repeated; the HR measurement will 
be repeated when below 50 bpm or above 100 bpm,  
 
major body systems via physical examination; general appearance, skin, neck 
(incl. thyroid), ENT (ears, nose, throat), lungs, heart, abdomen, lymph nodes (all 
major stations palpable), spleen, extremities, neurology, and other sites as 
applicable (i.e. symptom directed),  
 
safety blood samples for hematology, coagulation, serum chemistry, and 
urinalysis, to be performed and evaluated locally (as detailed in Section 7.3), and 
 
adverse events according to NCI-CTCAE v5.0.  
The following items should be assessed/done and documented at the visits specified below 
(see also Table 8): 
 
at visits V2, V4, V5, V7, V8, and V9, assessment of TSH, and free T4 for 
evaluation of thyroid function, and assessment of serum cortisol for evaluation 
of potential adrenal insufficiency; if the outcome is pathologic further testing 
should be considered per Section 7.3,  
 
for women of childbearing potential, a highly sensitive serum pregnancy test 
should be performed at visits V2, V4, V5, V7, V8, and V9,  
 
a 12-lead ECG should be recorded according to local site standard procedures at 
visits V7, and after this as medically indicated, 
 
a study kit to collect stools will be given to the patient at visit V4, and the patient 
will be asked to collect a stool sample any time prior to visit V5, 
 
tumor tissue to be collected at any time during study treatment when medically 
indicated procedure is done and it is possible to sample tissue, if the individual 
patient has consented to this procedure (the samples will be sent to a central 
laboratory for analyses as outlined in the objectives/endpoints of the trial; see 
Section 3). 
 
at visits V1, V2, V3, V4, V5, V6, V8, and V10 PBMC and serum will be sampled; 
at each sampling time the target is to collect 48 mL of blood for PBMC 
preparation, and 5 mL of blood for serum preparation - preparation/shipping 
procedures for the blood and serum samples will be detailed in the Laboratory 
Manual; a central laboratory will handle the immune response analyses,  
 
at visits V1, V2, V3, V4, V5, V6, V7, V8, V9, and V10 plasma will be sampled; 
at each sampling time the target is to collect 10 mL of blood for plasma 
preparation - preparation/shipping procedures for the samples will be detailed in 
the Laboratory Manual,  
 
at visits V6 and V10, planned CT scans of chest, abdomen, and pelvis, ctDNA 
testing, and testing of CEA (see Section 7.3 regarding possible additional serum 
tumor markers) should be done, 
 
and in addition, at visit V4, ctDNA testing, and testing of CEA (see Section 7.3 
regarding possible additional serum tumor markers) should be done. 


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Note, at suspicion of relapse of CRC adequate investigations per standard medical practice 
should be performed to verify, or not, the suspicion; any used method (e.g. CT, MRI, 
endoscopy, etc.) and outcome should be documented in the eCRF throughout the trial. 
7.5 
Post-treatment safety visits 
Thirty days (-0/+4 days) after the last administration of trial treatment, a safety visit (Vd30) 
should be done for all patients, in addition the Vd100 should be held 100 days after the last 
treatment with nivolumab. The visits should if possible be held as planned, but there are 
patient individual circumstances that might prevent a personal visit to the site, e.g. the patient 
might be in medical care at another hospital and in a medical status not amenable to 
transportation. In such cases, the Investigator should try collecting as much of the normally 
collected information as possible from the sources available.   
The items to be assessed/collected at visit Vd30 and Vd100 are (see also Table 8): 
 
concomitant medications/treatments,  
 
weight, and ECOG performance status (see Section 12.1), 
 
vital signs including temperature, blood pressure (BP), and heart rate (HR) (BP 
and HR will be measured according to local standard practice); if systolic BP is 
below 100 mmHg or above 150 mmHg, and/or diastolic BP is below 50 mmHg 
or above 90 mmHg, the measurement will be repeated; the HR measurement will 
be repeated when below 50 bpm or above 100 bpm,  
 
major body systems via physical examination; general appearance, skin, neck 
(incl. thyroid), ENT (ears, nose, throat), lungs, heart, abdomen, lymph nodes (all 
major stations palpable), spleen, extremities, neurology, and other sites as 
applicable (i.e. symptom directed),  
 
safety blood samples for hematology, coagulation, serum chemistry, urinalysis, 
TSH, free T4, serum cortisol, to be performed and evaluated locally (as detailed 
in Section 7.3),  
 
a 12-lead electrocardiogram (ECG) will be recorded according to local site 
standard procedures,  
 
for women of childbearing potential, a highly sensitive serum pregnancy test 
should be performed, and 
 
adverse events according to NCI-CTCAE v5.0.  
7.6 
Visits during post-treatment follow-up 
7.6.1 
Post-treatment visits before determination of relapsed disease 
Patients who have permanently stopped all study treatment without having had confirmation 
of relapsed disease will be followed every 3 months +/- 7 days, starting 3 months after V10 
(W27). 
At these timepoints the following is to be done (see also Table 8):  
 after week 27, CT scanning is to be done every 3-6 months per standard of care for 
the patient's stage of CRC disease (See Section 1.1 regarding recommendations for 
surveillance after curative treatments); collection of information from a protocol 
perspective should be done until scan documented relapse of disease (or until other 


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anti-cancer treatment would be started before confirmed progressive disease), or for 
a maximum of 3 years from first study treatment in the individual patient, 
 after week 27, continue ctDNA testing per local standard of care; collection of 
information from a protocol perspective should be done until scan documented 
relapse of disease (or until other anti-cancer treatment would be started before 
confirmed progressive disease), or for a maximum of 3 years from first study 
treatment,  
 PBMC and serum will be sampled every 3 months (+/- 7 days) until ctDNA test 
showing positive result, or scan documented relapse (or until other anti-cancer 
treatment would be started before confirmed progressive disease), or for a maximum 
of 3 years from first study treatment; at each sampling time the target is to collect 48 
mL of blood for PBMC preparation, and 5 mL of blood for serum preparation - 
preparation/shipping procedures for the blood and serum samples will be detailed in 
the Laboratory Manual; a central laboratory will handle the immune response 
analyses,  
 plasma will be sampled every 3 months (+/- 7 days) until ctDNA test showing 
positive result, or scan documented relapse (or until other anti-cancer treatment 
would be started before confirmed progressive disease), or for a maximum of 3 years 
from first study treatment; at each sampling time the target is to collect 10 mL of 
blood for plasma preparation - preparation/shipping procedures for the samples will 
be detailed in the Laboratory Manual,  
 after week 27, serum CEA to be tested (local lab) every 3 months (+/- 7 days) until 
ctDNA test showing positive result, or scan documented relapse (or until other anti-
cancer treatment would be started before confirmed progressive disease), or for a 
maximum of 3 years from first study treatment; note, if an individual patient earlier 
during treatment of his/her disease has shown a positive result for another serum 
measurable tumor marker than CEA, this tumor marker should be followed in 
addition to CEA, 
 documentation of safety; after the Vd30 and Vd100 post-treatment visits for safety 
assessment have been made, only SAEs with plausible causal relationship with trial 
treatment will be documented (in addition, in case of death, an as precise cause of 
death as possible is going to be documented),  
 if applicable, for female patients of childbearing potential, a serum pregnancy test 
will continue to be done on a monthly basis until 6 months after study treatments 
have been stopped, if not a new anti-tumor treatment has been initiated earlier in 
which case the study related testing will be terminated at the time of start of new anti-
tumor treatment, and 
 if applicable, collection of information regarding further anti-cancer treatments. 
Note, at suspicion of relapse of CRC adequate investigations per standard medical practice 
should be performed to verify, or not, the suspicion; any used method (e.g. CT, MRI, 
endoscopy, etc.) and outcome should be documented in the eCRF throughout the trial. 
7.6.2 
Post-treatment visits after determination of relapsed disease 
Patients who have permanently stopped all study treatment and have had confirmation of 
relapsed disease will be followed every 3 months +/- 7 days, starting 3 months after the time 
of determination of relapsed disease. 
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At these timepoints the following is to be done (see also Table 8):  
 assessment of survival status (if applicable, date of death, and cause of death, can be 
collected from local official registers or similar sources, e.g. records from other 
hospitals etc.), 
 documentation of safety; after the Vd30 and Vd100 post-treatment visits for safety 
assessment have been made, only SAEs with plausible causal relationship with trial 
treatment will be documented (in addition, in case of death, an as precise cause of 
death as possible is going to be documented),  
 if applicable, for female patients of childbearing potential, a serum pregnancy test 
will continue to be done on a monthly basis until 6 months after study treatments 
have been stopped, if not a new anti-tumor treatment has been initiated earlier in 
which case the study related testing will be terminated at the time of start of new anti-
tumor treatment, and 
 collection of information regarding further anti-cancer treatments.  
7.7 
Safety assessments 
Safety and tolerability of EO2040 in combination with nivolumab will be assessed by 
incidences of AEs, TEAEs, SAEs, deaths, reasons for treatment discontinuation/delays, and 
laboratory abnormalities. Adverse events will be categorized by their system organ class 
(SOC) and preferred term (PT) using the current MedDRA version and graded according to 
NCI-CTCAE v5.0 [100]. 
The trial safety setup is described in Section 4.3, the specific safety parameters to be 
monitored/assessed are outlined in Section 7, and safety monitoring procedures of the trial 
are described in Section 8. The timing of assessments is also described in Table 8. 
Clinical laboratory analyses as described in Section 7 will be performed by a local 
laboratory. More frequent evaluations may be performed at the Investigator’s discretion if 
medically indicated; results of such evaluations should also be recorded in the eCRF. Normal 
ranges for all utilized local laboratories should be provided by the sites to the Sponsor.  
7.8 
Efficacy assessments 
Response to therapy will be measured with a standard-of-care, CLIA certified ctDNA assay 
at the evaluation times specified in Table 8.  
Identification of somatic mutations in the blood will be confirmed by sequencing of the 
resected tumor using a CLIA-certified next-generation sequencing panel. Clearance of 
ctDNA will be characterized by the disappearance of all somatic mutations identified in the 
blood, as well as no appearance of any additional new somatic mutations.  
The primary endpoints will enumerate patients who have clearance of ctDNA, AND do not 
have any radiographic evidence of recurrence at 6 months.  
Secondary endpoints include assessments of tumor recurrence (the word relapse is used 
interchangeably with recurrence) via scanning (preferably CT, but MRI is accepted if local 
SOC; in any case method to be used as consistently as possible in an individual patient 
throughout the study) or other evaluations such as endoscopy. 
The best overall response is the best response recorded from the start of the treatment until 
disease recurrence.  The patient's best response assignment will depend on the achievement 
of both ctDNA and imaging criteria (see Table 9).  


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Table 9 : Assessment of best overall response  
Somatic ctDNA MT 
Imaging 
Protocol Response 
Cleared 
NED 
Responder 
Any 
Recurrence 
Non-responder 
Detectable 
Any 
Non-responder 
Not evaluated 
NED or Not evaluated 
Non-evaluable* 
Unequivocal PD 
Yes or No 
Non-responder 
Abbreviations: MT = mutation; NED = no evidence of disease; PD = progressive disease 
* If in FAS and the patient had important protocol deviations, the patient might be replaced after an 
assessment of the IDMC, and decision by the Sponsor. If the patient is not replaced, the patient will 
conservatively be assessed as non-responder. 
 
7.9 
Primary immunological assessments 
7.9.1 
Cell mediated immunity and associated utilization of PBMCs 
Cell-mediated cytotoxicity is a key element in the proposed mechanism of action of the 
peptides composing EO2040. Results from various clinical trials, including different modes 
of immunizations, have shown the suitability of the IFN-γ ELISpot assay for monitoring 
T cell responses during immunizations. Selection of an ex-vivo monitoring technique that 
provides a good measure of immune reactivity is important in determining potential 
correlations between clinical and immunologic responsiveness to specific immunotherapy. 
Cellular immune response in this trial will be evaluated by utilizing PBMCs in IFN-γ 
ELISpot assays, and by intracellular cytokine staining, and multimer staining. Samples will 
be collected at screening and during the trial according to Table 8. 
An important exploratory endpoint of the trial is the assessment of the percentage of patients 
with shown immunogenicity (expansion of specific T cells comparing samples taken at 
baseline versus on treatment in an individual patient determining if the patient has a positive 
response to the immunization, or not) in relation to the peptides that compose EO2040. Cross 
reactivities with the corresponding nominal peptides from BIRC5 and FOXM1 will also be 
evaluated. 
As exploratory objectives of the trial, additional scientific information related to 
characterization of factors impacting antitumor immunity will be searched for with the 
intention to explore their influence on safety and/or efficacy parameters. Thus, PBMCs not 
utilized for immunogenicity measurements according to above might be used for RNA-
sequencing of bulk and single cells to characterize factors as, for instance, T cell receptor-
variation and immune status. In addition, collected PBMCs might also be used for 
assessment of further parameters related to induction of immunity, especially once an 
immune induction is indicated, e.g. cell killing potential, and tests related to antigen 
spreading. 
Preparation/shipping procedures for blood samples will be detailed in the Laboratory 
Manual; a central laboratory will handle the cellular immune response analyses. 


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7.9.2 
Correlations between immunogenicity and other trial outcome parameters  
As an exploratory objective, statistical assessments of correlations between immunogenicity 
(as measured by specific T cell responses) of the components of EO2040 (or their full-length 
counterparts), and efficacy and safety outcome parameters will be conducted. Cross 
reactivities shown for the nominal antigens BIRC5 and FOXM1 will also be explored in the 
same way. 
7.10 Assessments for other exploratory endpoints 
Exploratory endpoints are outlined in Section 3.2.3, beside what has already been described 
in Section 7.9, the following assessment types are going to be utilized: 
 immunohistochemistry (IHC) of tumor tissue samples (from baseline available "old" 
tumor material) for expression of BIRC5 and FOXM1, and immune response/resistance 
related markers (e.g. HLA-A2, PD-L1, CD3, CD4, CD8, granzyme, Ki67; additional 
markers might be added, for instance if immune-resistance mechanisms relevant for 
immunization approaches would become known during trial conduct), aiming at defining 
immune profiles of tumor-infiltrating lymphocytes, and expression of immune markers 
on tumor cells and in the tumor microenvironment via determination of percentages of 
positive cells for the different markers, will be done at a central lab; in addition, and as 
possible by available material, molecular markers (presence, expression levels) will also 
be determined, including but not limited to mutations, deletions, and/or amplifications, 
and necessary assessments for determination of cancer molecular subtypes (also to be 
done at a central lab), 
 assessment of percentage of patients with changes on treatment versus baseline in serum 
markers (e.g. CEA), tumor tissue (when available), peripheral blood (PBMC) cell 
populations/markers, specific T cell populations (by e.g. TCR sequence analyses), T 
regulatory cells, and myeloid-derived suppressor cells (MDSCs), and changes in immune 
related markers as PD-L1/L2 expression, will be made by FACS, and other techniques,  
 tumor mutational burden, and disease specific somatic and germline mutations related to 
CRC, in individual patients as measured by the total number of nonsynonymous 
mutations per coding area with whole exome sequencing of tumor samples, and exome 
sequencing for germline mutations in blood samples; assessments will be made at a 
central lab, 
 DNA sequencing for metagenomics to assess overall fecal microbiome diversity will be 
done at a central lab,  
 for characterization of factors impacting antitumor immunity and definition of individual 
patient tumor inflammatory signatures, potentially delineating patients with a higher 
likelihood of tumor response to treatment, may include measurements in serum of 
cytokines such as IL-6 and inflammatory markers such as C-reactive protein, whole 
exome sequencing of tumors, and single cell RNA-sequencing of PBMCs, and 
 characterization of changes in ctDNA allele fractions over time with therapy will be done 
at a central lab. 
For all parameters described above, when possible, the intention is to explore their influence 
on safety and efficacy outcome parameters of the patients in the trial. 
 
 
 


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8 
SAFETY MONITORING 
8.1 
Safety monitoring definitions 
8.1.1 
Adverse events 
International Council for Harmonization (ICH) guideline E2A defines an AE as “any 
untoward medical occurrence in a patient or clinical investigation subject administered a 
pharmaceutical product and which does not necessarily have to have a causal relationship 
with this treatment”.  
According to the FDA (21CFR312.32), an AE means any untoward medical occurrence 
associated with the use of a drug in humans, whether, or not, considered drug related. 
An AE can therefore be any unfavorable and unintended sign (e.g. tachycardia, enlarged 
liver), symptom (e.g. nausea, chest pain), abnormal result of an investigation (e.g. 
laboratory finding), or disease temporarily associated with the use of a medicinal product, 
whether or not considered related to the medicinal product. 
8.1.2 
Serious Adverse events 
A serious adverse event (SAE) is any untoward medical occurrence that at any dose fulfills 
one or more of the following criteria: 
 results in death, 
 is immediately life-threatening, 
o the term “life-threatening” in the definition of “serious” refers to an event 
in which the patient was at immediate 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, 
 results in persistent or significant disability/incapacity, 
o disability is defined as a substantial disruption in a person’s ability to 
conduct normal life functions (i.e. the AE resulted in a significant, persistent, 
or permanent change, impairment, damage, or disruption in the patient’s 
bodily function/structure, physical activities, or quality of life), 
 is a congenital anomaly/birth defect, 
 requires inpatient hospitalization or prolongation of existing hospitalization, and 
o a hospitalization is defined as an inpatient overnight stay, but this can be 
shorter than 24 hours, 
 is another medically significant event defined as an event that may not be 
immediately life-threatening or result in death or hospitalization but may jeopardize 
the patient or may require intervention to prevent any of the above listed outcomes. 
8.1.2.1 
Excluded events 
Hospitalization for the following reasons will not be regarded as serious (i.e. will not be 
immediately reportable): 
 routine treatment or monitoring of the disease under study, including hospitalization 
due to trial-related procedures, not associated with any deterioration of the patient’s 
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 elective or pre-planned treatment (before signing the ICF) for a pre-existing 
condition that is unrelated to the disease under study and has not worsened since 
signing the ICF,  
 treatment on an emergency outpatient basis for an event not fulfilling any of the 
definitions for an SAE and not resulting in hospital admission, 
 underlying disease progression (the CRC which is the basis for the patient enrollment 
in the trial) and events which are unequivocally (without doubt) related to the disease 
progression, and 
 social reasons, respite care, and in the absence of a medical condition. 
In addition, as also outlined in Section 8.5.1, progression of the underlying disease or an AE 
unequivocally (without doubt) related to the progression of the underlying disease, 
regardless of its outcome or seriousness criteria, does not need to be reported as a SAE. 
However, the medical conditions and underlying diseases associated with disease 
progression needs to be captured on the AE eCRF page (as a non-serious AE). An SAE must 
be reported only if there are clinical symptoms/signs that cannot be without doubt associated 
with the progression of the underlying disease. 
8.1.3 
Suspected Unexpected Serious Adverse Reactions (SUSARs) 
SUSARs are serious events that are not listed in the Reference Safety Information section of 
the EO2040 Investigator Brochure and are related to the study drug. Suspected adverse 
reaction means any AE for which there is a reasonable possibility that the study drug caused 
the AE. For the purposes of Investigational New Drug safety reporting, “reasonable 
possibility” means there is evidence to suggest a causal relationship between the study drug 
and the AE.  
8.1.4 
Severity/intensity versus seriousness 
ICH E2A: The term “severe” is often used to describe the intensity (severity) of a specific 
event (as mild, moderate, or severe myocardial infarction); the event itself, however, may be 
of a relatively minor medical significance (such as a severe headache). This is not the same 
as “serious”, which is based on patient/event outcome or action criteria usually associated 
with events that pose a threat to a patient’s life or functioning. Seriousness (not severity) 
serves as a guide for defining regulatory reporting obligations. 
8.2 
Pregnancies 
If not surgically sterile, female patients of childbearing potential age must use highly 
effective contraception from signing the ICF through 6 months after the last treatment dose 
administered (see Section 5.1). 
If not surgically sterile, male with female partner of childbearing potential age must use 
condom from signing the ICF through 6 months after the last treatment dose administered. 
Males must also ensure that their partners of childbearing potential use highly effective 
contraception (see Section 5.1). 
Pregnancy is not considered an AE unless it meets any criteria for becoming serious (see 
Section 8.1.2). However, it is the responsibility of the Investigator or their designee to report 
immediately any pregnancy in a patient or the patient’s sexual partner that occurs during the 


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study (see Section 8.5.3 regarding reporting procures). All patients who become pregnant 
must immediately discontinue the study drug and be withdrawn from study treatment.  
The Investigator should make every effort to follow the patient until completion of the 
pregnancy and provide the corresponding information by the mean of a SAE/Pregnancy form 
within 2 weeks from awareness (see Section 8.5.3). The Sponsor Safety Group will 
periodically request from the investigator targeted follow-up information using a specific 
Pregnancy Follow-up form. If the events during pregnancy and/or outcome of pregnancy 
(i.e. complications regarding mother/baby) meet the criteria for classification of an SAE, the 
Investigator must follow the procedures for SAE reporting. 
8.3 
Safety monitoring periods 
8.3.1 
Reporting 
In patients exposed to study treatment, the following must be reported from the date of the 
patient’s signing the ICF until 30 days after the final administration of study treatment: 
 AEs regardless of their causal relationship to study treatment, 
 SAEs without a plausible causal relationship to study treatment, and 
 pregnancy of the patient or the patient’s sexual partner. 
 
In addition, for consistency with nivolumab clinical trial protocols as advised by Bristol-
Myers Squibb (marketing authorization holder for nivolumab), report all non-serious adverse 
events (not only those deemed to be treatment-related) continuously during nivolumab 
treatment periods and for a minimum of 100 days following discontinuation of nivolumab 
treatment. 
SAEs with a plausible causal relationship to study treatment must be reported from the date 
of the patient’s signing the ICF until indefinitely (regardless of the time elapsed from the 
final study treatment administration). 
In patients not exposed to study treatment, the reporting period for AEs (including SAEs 
regardless of causal relationship) starts with signature of the ICF and ends: 
 the date of the Investigators assessment of "screen failure"-status (see Section 5.3.2), 
or 
 the date of the patient’s consent withdrawal. 
8.3.2 
Follow-up 
The Investigator assesses at each visit (or more frequently, if necessary) if there are any new 
AEs or any changes in AE diagnosis, severity, suspected causal relationship to clinical trial 
medication/procedure, interventions required to treat the event, and AE outcome.  
An AE causally not related to study treatment is monitored (followed-up) until resolution, 
stabilization (becoming a permanent condition), or end of the clinical trial. Clinically 
relevant laboratory abnormalities will be followed up until they return to normal or become 
stabilized (permanent condition). 
Any AE with a plausible causal relationship to study treatment as well as all SAEs 
(regardless of their causal relationship) will be followed up until the event has resolved or 
stabilized (permanent condition). 


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Pregnancies will be monitored by the Investigator to determine the outcome, including 
spontaneous abortion or voluntary termination, birth details, and the presence or absence of 
any birth defects, congenital abnormalities, or maternal and newborn complications. Every 
infant should be followed up for 2 months after delivery. 
8.4 
Assessing adverse events 
Information about adverse reactions (causally related events) already known for EO2040 
monotherapy, or EO2040 in combination with nivolumab, can be found in the current 
version of the EO2040 IB or will be communicated between IB updates in the form of “Dear-
Investigator Letter”. 
Information about adverse reactions (causally related events) already known for nivolumab 
can be found in the labelling documents for nivolumab [90, 91]. 
8.4.1 
Causality 
The Investigator needs to assess the causal relationship of any AE in relation to the study 
treatments. In context of the study treatments, EO2040 and nivolumab are given in 
combination in Cohort 1, it is most of the times in principle not possible to make causality 
assessments for each component by itself.  
However, there will be a possibility in the eCRF to record causality for EO2040 and 
nivolumab as separate components for each AE, or to select to make an overall assessment 
for both study treatments together.  
The causality assessment is based on the Investigator’s clinical judgment taking into 
consideration all relevant information available at the time of AE reporting including: 
 temporal association of the event onset with administration of the medication, 
 known type of reaction for the administered compounds, 
 disappearance or abating of symptoms when the compound(s) is discontinued, or 
withheld, 
 reappearance of symptoms when the compound is re-administered, 
 event may or may not be caused by the patient’s health condition, 
 presence of risks or factors not related to trial intervention that are known to be associated 
with the occurrence of the event. 
Causal relationship of all AEs will be classified as follows: 
 Not suspected: it is not plausible that the AE is caused by the medication(s) and a likely 
alternative explanation exists. There is no reasonable possibility of a causal relationship. 
 Suspected: it is plausible that the AE is caused by the medication(s). There is a 
reasonable possibility of a causal relationship.  
For the purposes of safety reporting, ‘reasonable possibility’ means there is evidence to 
suggest a causal relationship between the medication(s) and the AE. 
8.4.2 
Severity/intensity 
Severity or intensity of an AE should be assessed according to the NCI CTCAE v5.0 [100]; 
a grading scale is provided for AE terms displaying grades 1 through 5 with unique clinical 
descriptions of severity for each AE. Note, possible changes of the severity/intensity of 
adverse events should be monitored, and when applicable documented. 


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8.5 
Reporting by the investigational site 
8.5.1 
Adverse events 
Any AE (including SAEs), whether, or not considered to be causally related to the trial 
medication and regardless of its seriousness, must be described and recorded in the AE 
section of the patient’s eCRF on an ongoing basis. 
A pre-existing condition is a clinical condition (including a condition being treated) that is 
diagnosed before the patient signs the ICF and that is documented as part of the patient’s 
medical history; any clinically significant worsening of a pre-existing condition constitutes 
an AE. Also, any recurrence of a pre-existing condition is an AE. 
The questions concerning whether the condition existed before the start of the active phase 
of the study and whether it has increased in severity and/or frequency will be used to 
determine whether an event is a TEAE. An AE is considered to be treatment-emergent if it 
is not present when the active phase of the study begins and is not a chronic condition that 
is part of the patient’s medical history, or it is present at the start of the active phase of the 
study or as part of the patient’s medical history, but the severity or frequency increases 
during the active phase. The active phase of the study begins at the time of the first dose of 
the study drug. The active phase of the study ends at the post-treatment visit for safety 
assessment (see Table 8). 
A standardized question such as “Have you had any health problems since your last visit or 
since you were last questioned?” will be given by the Investigator or the investigational site 
personnel at each contact with the patient. 
Progression of the underlying disease or an AE unequivocally (without doubt) related to the 
progression of the underlying disease, regardless of its outcome or seriousness criteria, does 
not need to be reported as an AE/SAE. However, the medical conditions and underlying 
diseases associated with disease progression needs to be captured on the AE eCRF page (as 
a non-serious AE). An SAE must be reported only if there are clinical symptoms/signs that 
cannot be without doubt associated with the progression of the underlying disease. 
Whenever possible, a diagnosis rather than symptoms should be provided (e.g. anemia 
instead of low Hb). 
Abnormal laboratory and other abnormal examinations (e.g. at physical examination) should 
not be reported as AEs, unless they are associated with clinical signs/symptoms, require 
medical intervention/therapy (e.g. transfusion due to low Hb), require a change in trial 
medication (e.g. temporary interruption of treatment, or definitive treatment 
discontinuation), or are otherwise considered clinically relevant by the Investigator. 
Clinically relevant laboratory, or examination abnormalities will be followed up until they 
return to normal or become stabilized (permanent condition).  
If a clinically significant laboratory abnormality is a sign of a disease or syndrome (e.g. ALP 
and bilirubin 5 times the ULN associated with cholecystitis), only the diagnosis (i.e. 
cholecystitis) should be recorded on the AE eCRF page. 
If a clinically significant laboratory abnormality is not a sign of a disease or syndrome, the 
abnormality itself should be recorded on the AE eCRF page, along with a descriptor 
indicating if the test result is above or below the normal range (e.g. “elevated potassium,” as 
opposed to “abnormal potassium”). If the laboratory abnormality can be characterized by a 
precise clinical term per standard definitions, the clinical term should be recorded as the AE. 


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For example, an elevated serum potassium level of 7.0 mEq/L should be recorded as 
“hyperkalemia.” 
Physical examination findings will be compared with the baseline status and any clinically 
significant change, as assessed by the Investigator, should be documented as an AE. 
A surgical procedure is not an AE but a therapeutic measure for a condition that necessitates 
surgery. Therefore, the condition for which the surgery is required should be reported as an 
AE.  
Any pre-planned surgery (i.e. planned before signature of the ICF) or other intervention 
permitted by the protocol and the condition leading to that measure are not AEs. In such 
cases, the underlying condition needs to be documented in the patient’s medical history. 
Death itself is an outcome of an event, which needs to be described and reported using 
medical terminology. Information about death will be captured on the respective eCRF page 
along with relevant details (date of death, immediate and underlying causes of death). 
8.5.1.1 
Adverse Events Associated with an Overdose or Error in Treatment 
Administration 
Study drug overdose is the accidental, or intentional, use of the drug in an amount higher 
than the dose being studied. An overdose, or incorrect administration, of study drug is not 
an AE unless it results in untoward medical effects. 
Any study drug overdose or incorrect administration of study drug should be noted on the 
Study Drug Administration eCRF page. 
All AEs associated with an overdose or incorrect administration of study drug should be 
recorded on the AE eCRF page. If the associated AE fulfils serious criteria, the event should 
be reported to the Sponsor immediately (i.e., no more than 24 hours after learning of the 
event; see Section 8.5.3 for reporting instructions). 
8.5.2 
Documenting in the eCRF 
The reported term should be a medical diagnosis or sign/symptom of the event, not a 
procedure. Each symptom in a constellation of symptoms should be listed separately if the 
Investigator has not made a preliminary/tentative summary diagnosis. 
Fluctuations or re-occurrences of a condition, which are considered normal for the patient 
and are recorded in patient’s eCRF medical history, do not need to be reported as an AE. 
However, if the condition deteriorates during the trial it needs to be captured as an AE. 
If the same AE occurs repeatedly, it must be assessed and documented separately each time 
if there are "AE-free" time periods between the AEs. 
If possible, each AE should be evaluated to determine: 
 event term or a description of the AE in medical terms (not as reported by the patient), 
 severity grade as assessed by the Investigator (1 - 5 per NCI CTCAE v5.0), 
 its causal relationship to study treatment(s) as assessed by the Investigator (suspected; 
not suspected), 
 event duration, including onset date and end date, 
 action taken with study treatments due to the reported event (dose not changed; drug 
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 other action taken (no action taken; medication required; surgical intervention required; 
other), 
 event seriousness (non-serious or serious AE), 
 event outcome (recovered/resolved, recovered/resolved with sequelae, not recovered/not 
resolved, fatal, unknown), and 
 disease progression-related event (yes, no). 
8.5.3 
Immediately reportable events - Serious Adverse Events & Pregnancies 
The Investigator or designated investigational site staff must immediately (within 24 hours 
of awareness at the latest) notify/report to pharmacovigilance at the Sponsor, i.e. Enterome 
(handled by Stragen Services SAS, 19 rue Jacqueline Auriol, 69008 Lyon, France; referred 
to as the Safety Group), any initial or medically relevant follow-up information about SAEs, 
or pregnancies (see Section 8.2). 
All immediate reports from the investigational site to the Safety Group (i.e. SAEs and 
pregnancy reports) must also be recorded in the site’s source documentation. 
The primary mode of reporting a SAE is based on entering the appropriate and complete 
information into the clinical database via the SAE reporting form and submitting the SAE 
form within the electronic data capture (EDC) system (eCRF-system) to the trial Safety 
Group within 24 hours of obtaining knowledge of the event. 
The SAE Form (in the eCRF) is to collect data surrounding the event (e.g. the nature of the 
symptom[s], time of onset in relation to initiation of therapy, duration, intensity, and 
whether, or not therapy was interrupted or discontinued). The Investigator’s assessment of 
the probable cause of the event will also be included. In addition, relevant medical history, 
concomitant medications, laboratory and diagnostic test reports, and procedures as well as 
all pertinent medical information related to the event will also be collected. Once the AE 
page has been updated in the eCRF for an SAE by the site, the site will submit it to the safety 
group within the system. Then an alert from the clinical database will be initiated and sent 
to the Safety Group. The Safety Group will generate the SAE report, a PDF extracted from 
the clinical database, and send to the Sponsor. 
The Safety Group will generate SAE queries requesting missing information, correction of 
implausible, etc., and add the queries directly in the clinical database. It is the Investigator’s 
responsibility to be diligent in providing the answer as soon as it is available by entry of the 
corrections in the eCRF and submission of the follow-up SAE report to the Safety Group. 
In case of technical or any other reason the SAE reporting cannot be carried out via the EDC 
system per above, the Investigator (or designated investigational site staff) will complete a 
paper SAE form and send to the Safety Group as outlined below. The paper SAE form will 
be provided to the sites prior to start of the study. 
Note, SAE report forms must be completed in English. 
When a paper form is completed, follow-up information should not be reported on the same 
form that was used for the initial reporting, but using a new form filled in only with the new 
information. Changes/completions need to be done in a Good Clinical Practice (GCP)-
compliant manner (i.e. dated and initialized). Originals of the report forms must be kept in 
the site study file (if certified electronic signature is available at site, electronic copy of the 
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Initial reports of SAEs should never be left on telephone voicemails. In case of using the 
paper SAE form, please always fax or e-mail the SAE report, and follow-up with a telephone 
call if needed. 
Serious AEs to be reported to the Enterome/Stragen Safety Group: 
 
Facsimile:  
 
+33 (0) 4 78 42 55 71 
 
E-mail:  
 
EnteromePV@stragen.fr 
In case of urgent questions regarding SAE reporting please call: 
 
Enterome Medical Monitor  
+33 (0) 6 14 31 07 59 
 
Stragen Services 
 
 
+33 (0) 4 78 42 95 26 
The initial Pregnancy Data Collection form should be handled in the same way as an initial 
SAE report, i.e. via the EDC system, or via the specific paper form (latter only in case of 
technical issues with the EDC).  
Follow-up information is to be sent within the same timelines using the same modes of 
reporting as outlined above.  
All sites will follow their institutional requirements for submission of SAEs to their 
IRBs/IECs. 
The processing and reporting of all relevant SAEs to authorities will be done by the Sponsor 
or Sponsor’s designee according to all applicable rules/regulations, including reporting of 
unexpected serious adverse events to the USA FDA according to 21 CFR 312.32. The 
Sponsor or designee will inform all investigational sites about reported relevant events 
according to applicable regulations. 
Note for France (valid per March-2022, i.e. future changes might take place): fatal or life 
threatening SUSARs will be reported to ANSM without delay, i.e. within 1 working day, 
with additional relevant information provided in the following 8 calendar days. 
Note, in addition to the reporting of SAEs per above, the trial includes immediate reporting 
per below for all related Grade ≥ adverse events (see also Section 4.3): 
 All related Grade ≥ 3 adverse events should be reported within 24 hours to the Sponsor 
via the Medical Monitor (reporting can be via the medical monitoring e-mail 
[medicalmonitoring-crc@enterome.com] or the medical monitoring telephone [+33 
(0) 6 14 31 07 59]), and ALSO be reported as an SAE if it meets such criteria (see 
Section 8.5.3). Note, if the eCRF is filled out with the related Grade ≥ 3 adverse event 
within 24 hours this is also accepted as information to the Sponsor since there will be 
a special alert system implemented for such recordings in the eCRF.  
8.5.3.1 
SAE minimum notification/reporting requirements 
The following information must be provided for a valid notification/report: 
 identification of the notifying/reporting person (e.g. name of the reporter), 
 identification of the patient (e.g. patient trial identification number), 
 concerned IMP and/or clinical trial (e.g. EO2040, trial EOCRC1-22), 
 reason for notification/reporting (i.e. SAE, or pregnancy), and 
 event term. 


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In addition, providing the assessment of the causal relationship is necessary for 
comprehensive evaluation by the Sponsor. 
8.6 
Independent Data Monitoring Committee (IDMC) 
The IDMC will serve as an external monitoring group for the study. The primary role of the 
IDMC will be to examine the safety and tolerability of study participants throughout the 
duration of the study. The IDMC will be created to further protect the rights, safety, and 
well-being of patients who will be participating in the trial by monitoring their progress and 
results.  
The IDMC will examine the safety data on an ongoing basis regarding any related Grade ≥ 
4 events, and regarding related Grade 3 events if deemed necessary by the investigator 
responsible for the patient in question or Sponsor. In addition, a pre-planned safety 
assessment will take place at the end of Cohort 1 (see Section 9.3). 
In relation to the efficacy assessment for Cohort 1 (and linked possible transition to Cohort 
2A), the IDMC will also oversee and validate the efficacy assessment as performed by the 
Sponsor (or designee). 
The IDMC will be consulted by the Sponsor in relation to possible replacement of patients 
in FAS (see Section 5.3.8). 
The IDMC members, including a minimum of three qualified persons, are to be international 
hematology/oncology experts, or qualified clinical trial statistician (there is no demand for 
statistical expertise among the IDMC members for this trial), who are not in other ways than 
via the IDMC involved in the current trial. Details of the IDMC processes and procedures is 
outlined in a separate IDMC Charter. 
 
 
 
 
 


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9 
STATISTICAL EVALUATION  
9.1 
Analysis populations 
The All-Patient (AP) population will consist of any patient who signed informed consent 
including screen-failures. 
The Full Analysis Set (FAS) will consist of patients who received at least one dose of 
EO2040 and for whom no important protocol deviations occurred that would compromise 
the evaluation of efficacy. 
The Safety Set (SS) will consist of patients who received at least one dose of study treatment. 
The Per-Protocol (PP) population will consist of patients who received at least 5 doses of 
EO2040 (and when applicable associated nivolumab), for whom no important protocol 
deviations occurred, and who have at least one evaluable post-screening ctDNA and tumor 
imaging. 
Efficacy will be analyzed using both the FAS and PP populations. Safety will be analyzed 
using the SS, however applicable adverse events for the AP population will be reported in 
by-patient listings (e.g. events during screening). 
9.2 
Statistical methods 
All data collected in this trial will be reported by patient data listings, summary tables, and 
figures for all demographic and baseline characteristics, medical history, efficacy, and safety 
variables. Proportions and the denominators will be provided to summarize response, 
toxicity, and other categorical variables.  
The response rate R6 will be estimated using all patients in the FAS with response at 6 
months in the numerator and all patients in the FAS in the denominator; and the analysis will 
be repeated based on all patients in the PP population. Two-sided exact 95% confidence 
intervals will be provided. A treatment is defined to be successful, if the one-sided null 
hypothesis of R6 being smaller or equal to 5% (H0: P ≤ 5%) can be rejected on a one-sided 
2.5% significance level.  DFS and overall survival will be analyzed using the method of 
Kaplan and Meier and presented with two-sided 95% confidence bands. 
Statistical assessments of correlations between e.g. immunogenicity parameters (see Section 
3.2.3, and Section 7.9) and efficacy and safety outcome parameters will be conducted as 
described in the Statistical Analysis Plan (SAP).  
The SAP will provide all details with respect to patients being analyzed for all endpoints. 
Relevant subgroups based on available data and compliance to protocol procedures might 
be defined. 
Statistical tests may be planned in the SAP or performed as needed. The importance of the 
tests does not reside in making conclusions on statistical relevance of differences, but rather 
identification of possible results that could be worth further exploration. 
9.3 
Interim analyses 
The nature of the study, i.e. early exploratory development trial aiming at generating as much 
knowledge as possible before potential decisions related to further development of EO2040, 
makes it important to assess especially safety, but also efficacy and possible biomarkers, on 
an ongoing basis during trial conduct. Exploratory preliminary data from the trial might be 
utilized in relation to e.g. scientific discussions and presentations to facilitate input regarding 
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A definitive interim analysis will be performed at the end of Cohort 1 regarding the primary 
objective, secondary objectives, and exploratory objectives. The timing of the analysis is to 
be linked to the time when the last patient to be analyzed (among the patients in Cohort 1 
who have not relapsed at an earlier timepoint) has had his/her week 27 examinations. 
9.4 
Determination of sample size & primary analysis  
This is an early development, open-label, exploratory, trial to include patients with CRC 
who have early disease which today is without an established standard of care, but still in a 
relevantly early stage of the overall disease history that there is a potential chance to 
significantly delay recurrence/progression with an efficacious therapy. Thus, a balance needs 
to be struck between a size to find a relevant signal, and the importance to not expose too 
many patients to a possibly non-efficacious therapy.  
Considering prior attempts in similar situations, the trial will include 17 patients per cohort. 
Utilizing FAS as the population for the primary analysis, the basic assumption is that there 
will be no non-evaluable patients. However, if there would be any doubt if a patient would 
be included in the FAS, or not, the Sponsor will consult the IDMC and if recommended the 
patient might be replaced. 
A conservative estimate is that the accrual rate will be approximately 1.5 patients per month 
considering the outlined inclusion/exclusion criteria (see Section 5.1 and Section 5.2). 
The primary objective of the trial is to determine the ctDNA clearance rate at 6 months (R6).  
The one-sided null hypothesis H0: R6 ≤ 5% will be tested against the alternative hypothesis 
HA: R6 ≥ 32.5%, using alpha = 0.025 and an exact binomial test. Assuming thus a 
spontaneous ctDNA clearance rate of 5%, and a true ctDNA clearance rate under study 
treatment being at least 32.5% (study treatment being EO2040 in combination with 
nivolumab or EO2040 monotherapy, with the possible addition of nivolumab at 3 months; 
Cohort 2A) 17 patients will provide a power of 85%.  
For the primary analysis, the ctDNA clearance rate will be estimated along with the 95% 
exact confidence interval. The efficacy of study treatment will also be assessed by 
performing a binomial test comparing the ctDNA clearance rate against the null hypothesis 
5%, at a one-sided alpha level of 0.025.  
If at least 4 out of 17 patients in the Full Analysis Set (FAS; patients who received at least 
one dose of EO2040 and for whom no important protocol deviations occurred) show 
response to study therapy in a cohort, the therapy will be declared successful. 
Assuming 4 of the 17 patients would achieve a response, the observed response rate would 
be 24% and the corresponding 95% exact confidence interval would be (7.1% to 50.4%).   
The opening of a Cohort 2A exploring EO2040 monotherapy (with the option of addition of 
nivolumab if ctDNA is not cleared at 3 months) is dependent on the outcome of Cohort 1; 
the main factor being the assessment of ctDNA clearance rate (which should be positive per 
above), but also DFS might be considered. If no continuation of exploration of EO2040 
would be seen as appropriate based on data from Cohort 1, there will be considerations 
regarding a protocol amendment to switch EO2040 to an alternative microbiome-derived 
vaccine.  
Based on the above, it is assumed that the current trial will include enough patients to provide 
safety and tolerability information, immunogenicity data, as well as preliminary efficacy 


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data in the selected treatment setting without demanding a too high (and long) patient 
recruitment and without exposing too many patients.  
 
10 
 ADMINISTRATIVE CONSIDERATIONS 
10.1  
Regulatory and ethical considerations 
This study will be conducted in accordance with the protocol and with the following: 
 consensus ethical principles derived from international guidelines including the 
Declaration of Helsinki and Council for International Organizations of Medical 
Sciences International Ethical Guidelines, 
 applicable International Conference on Harmonisation of Technical Requirements 
for Registration of Pharmaceutical for Human Use (ICH) Good Clinical Practice 
(GCP) Guidelines, and 
 applicable laws and regulations; approval will be obtained from the appropriate 
regulatory authorities before any site is initiated in a country. 
Before study start, each site Principal Investigator (PI) is required to sign a protocol signature 
page confirming his agreement to conduct the study in accordance with these documents and 
all of the instructions and procedures found in this protocol and to give access to all relevant 
data and records to clinical research associates (CRAs), auditors, the Sponsors quality 
assurance representatives, designated agents of the Sponsor, ECs, and regulatory authorities 
as required. 
The protocol, protocol amendments, Informed Consent Form (ICF), Investigator’s Brochure, 
and other relevant documents (e.g. advertisements) must be submitted to an Institutional 
Review Board (IRB)/Independent Ethics Committee (IEC) by the Investigator and reviewed 
and approved by the IRB/IEC before the study is initiated. 
The Investigator will be responsible for the following: 
 providing written summaries of the status of the study to the IRB/IEC annually or 
more frequently in accordance with the requirements, policies, and procedures 
established by the IRB/IEC, 
 notifying the IRB/IEC of serious adverse events (SAEs) or other significant safety 
findings as required by IRB/IEC procedures, and 
 providing oversight of the conduct of the study at the site and adherence to 
requirements of 21 Code of Federal Regulations (CFR), ICH guidelines, the 
IRB/IEC, European regulation 536/2014 for clinical studies (if applicable), and all 
other applicable local regulations. 
A signed and dated statement that the protocol and ICF have been approved by the ECs must 
be given to the Sponsor (or designee) before study initiation.  
10.2  
Finances and insurances 
Financing and insurance will be addressed in separate site agreements. 
For each participating patient, the Sponsor has taken out insurance covering the amount 
determined by respective national laws. All participating patients will be informed about the 
existence of the insurance in the patient informed consent. They have the right to review the 
terms and conditions of the insurance. 


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10.3  
Informed consent 
Written informed consent for the study will be obtained from all patients before any 
protocol-specific procedures are conducted. The ICF generated by the Sponsor or designee 
will be approved (along with the protocol) by the IRB/IEC. 
Information about the study will be given to the patient both verbally and in writing. The 
written patient information sheet will explain the objectives of the study and its potential risk 
and benefits. The patient should have adequate time to read the information sheet and to ask 
the Investigator any questions. The Investigator must be satisfied that the patient has 
understood the information provided before written consent is obtained. If there is any doubt 
as to whether the patient has understood the written and verbal information, the patient 
should not enter the study. 
If a patient agrees to participate, he/she will be asked to sign and date the study ICF, which 
will be retained by the Investigator. A copy of the signed ICF will be given to the patient. 
The informed consent process must be documented in the patient’s source documents. The 
original ICF must be retained by the Investigator and made available for inspection by the 
Study Monitor. 
10.4  
Future use of patient samples; sample traceability 
Following the completion of all study testing, the remaining tissue and blood/blood 
components may be used for future biomedical research (e.g. additional analysis on PBMC). 
This research will help to understand response against other tumor-associated antigens not 
included in the current peptide mix and possibly include DNA sequencing. If a patient 
requests destruction of his/her tissue and blood samples and the samples have not yet been 
de-identified, the Sponsor will destroy the samples under specific regulations and notify the 
Investigator in writing that the samples have been destroyed. Patients will be asked whether, 
or not they consent to the (optional) future use of patient samples. This consent will be 
documented separately, and patients will not be excluded from study participation if they do 
not consent to this future use of residual samples. 
Blood samples being collected and stored for research purposes within this study should be 
traceable as required by local regulations, or the clinical trial authorization, as applicable. 
The Sponsor and Investigator institutions should keep their parts of the traceability records 
accordingly.  
10.5  
Patient data protection 
Patients will be assigned a unique identifier and will not be identified by name in eCRFs, 
study-related forms, study reports, or any related publications. Patient and Investigator 
personal data will be treated in compliance with all applicable laws and regulations. In the 
event the study protocol, study report, or study data are included in a public registry, all 
identifiable information from individual patients or Investigators will be redacted according 
to applicable laws and regulations. 
The patient must be informed that his/her personal study-related data will be used by the 
Sponsor in accordance with local data protection law. The level of disclosure must also be 
explained to the patient. The patient must also be informed that his/her medical records may 
be examined by the Sponsor or Contract Research Organization (CRO) auditors, or other 
authorized personnel appointed by the Sponsor, and by inspectors from regulatory 
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10.6  
Site monitoring 
Before study initiation, at a site initiation visit or at an Investigator’s meeting, Sponsor 
personnel (or a designated contract research organization [CRO]) will review the protocol 
and eCRFs with the Investigators and their staff. During the study, the field monitor will 
visit the site regularly to check the completeness of patient records, the accuracy of filling 
of the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, and to 
ensure that the study drug is being stored, dispensed, and accounted for according to 
specifications. Key study personnel must be available to assist the field monitor during these 
visits.  
The Investigator must maintain source documents for each patient in the study recruited at 
the relevant site, consisting of case and visit notes (hospital or clinic medical records) 
containing demographic and medical information, laboratory data, ECGs, and the results of 
any other tests or assessments. All information on eCRFs must be traceable to these source 
documents in the patient’s file. The Investigator must also keep the original ICF signed by 
the patient (a signed copy is given to the patient).  
The Investigator must give the monitor access to all relevant source documents to confirm 
their consistency with the eCRF filling. The Sponsor monitoring standards require full 
verification for the presence of informed consent, adherence to the inclusion/exclusion 
criteria, documentation of SAEs, and the recording of data that will be used for all primary 
and safety variables. Additional checks of the consistency of the source data with the eCRFs 
are performed according to the study-specific monitoring plan. No information in source 
documents about the identity of the patients will be disclosed. 
10.7  
Data collection 
All individual, patient-specific study data will be entered into a 21 CFR Part 11-compliant 
electronic data capture (EDC) system on an eCRF in a timely fashion.  
The Sponsor, or designee, will supply the investigational site with access to a web-based 
EDC computer system. The designated Investigator site staff will not be given access to the 
EDC system until they have been trained. 
Access to the EDC system at the site, for vendors, at the Sponsor, and at the CRO is password 
protected. Study access is granted to site personnel only after they have been trained in the 
use of the EDC system by web-based training at the investigational site. 
The EDC system contains a system generated audit trial that captures any changes made to 
a data field, including who made the change, and the date and time it was made. This 
information is available at the Investigator’s site, and at the Sponsor (and CRO when 
applicable). 
Data will be entered into the study database by the Investigator/Study Coordinator at each 
site. Entries made in the EDC screens should be completed within 5 days of the patient’s 
study visit (see Section 8.5 regarding timelines for immediately reportable events) and must 
be supported by source documents maintained for all patients enrolled in the study. 
Data generated from external sources (e.g. central laboratory) and transmitted to the Sponsor 
or designee electronically might be integrated with the patient’s eCRF data in accordance 
with the Data Management Plan. 
An eCRF must be completed for each patient who signs an ICF and undergoes any 
pre-screening or screening procedures, according to the eCRF completion instructions. 


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Automatic validation programs check for data discrepancies in the eCRFs and, by generating 
appropriate error messages, allow modification or verification of the entered data by the 
Investigator staff before transfer of data to the Sponsor (or a designated CRO).  
Sponsor personnel (or a designated CRO) will review the eCRFs entered by site staff for 
completeness and accuracy (including as applicable verification against source documents) 
and instruct the site personnel to make any required corrections or additions. System or 
manually generated queries are raised to the investigational site using the study EDC system. 
Designated Investigator/site staff is required to respond to the queries and make any 
necessary changes to the data.  
The Investigator will sign and date the eCRF via the EDC system’s electronic signature 
procedure. These signatures will indicate that the Investigator reviewed and approved the 
data on the eCRF, the data queries, and the site notifications. 
At the conclusion of the study, the occurrence of any protocol violations will be determined 
(guidance can be found in Section 12.3).  
After these actions have been completed and the database has been declared to be complete 
and accurate, it will be locked. 
After database lock, the Investigator will receive a CD-ROM of the patient data for archiving 
at the investigational site. 
10.8  
Protocol amendments 
Any substantial amendments in the research protocol during the period, for which the 
IEC/IRB approval had already been given, will not be initiated without submission of an 
amendment for IEC/IRB review and approval. 
These requirements for approval will in no way prevent any immediate action from being 
taken by the Investigator in the interest of preserving the safety of all patients included in 
the trial. 
10.9  
Protocol deviations and violations 
A protocol deviation is any departure from the defined procedures and treatment plans as 
outlined in the protocol version submitted and previously approved by the IEC/IRB. Protocol 
deviations have the potential to place participants at risk and can also undermine the 
scientific integrity of the study, thus jeopardizing the justification for the research. Protocol 
deviations are unplanned and unintentional events. 
Important protocol deviations, such as significant non-compliance or other serious 
unforeseen deviations deemed to invalidate the data collected in lieu of the purpose of the 
study will lead to exclusion of data from analysis. 
All decisions regarding the type of deviations will be made prior to commencing the final 
analysis on the finally locked database. A listing of patients with protocol deviations will be 
maintained by the Sponsor (or designee) and a listing of protocol violations (see Section 
12.3) will be presented in the final study report. 
Investigators shall apply due diligence to avoid protocol deviations. If the Investigator feels 
a protocol deviation would improve the conduct of the study, this must be considered a 
protocol amendment, and unless such an amendment is agreed upon by the Sponsor and 
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The Investigator will report protocol deviations to their IEC/IRB per institutional reporting 
requirements. 
10.10 
Change in investigator 
If any Investigator retires, relocates, or otherwise withdraws from conducting the study, the 
responsibility for maintaining records may be transferred to the Sponsor or designee, IRB, 
or another Investigator. The Sponsor or designee must be notified of and agree to the change. 
Regulatory agencies will be notified with the appropriate documentation. 
10.11 
Clinical study report 
A clinical study report will be prepared following the completion of the study. The Global 
Coordinating Investigator should if required by local law/regulations be the Investigator who 
should review and sign the study report. 
10.12 
Confidentiality/disclosure 
All information provided regarding the study, as well as all information collected and/or 
documented during the study, will be regarded as confidential. The Investigator (or designee) 
agrees not to disclose such information in any way without prior written permission from 
the Sponsor. 
10.13 
Record retention 
Essential documents must be retained for longer than 5 years after completion of the study, 
2 years after the final marketing authorization in an ICH region or until at least 2 years have 
elapsed since the discontinuation of clinical development of the study drug. If it becomes 
necessary for the Sponsor or a relevant competent authority to review any documentation 
relating to the study, the Investigator must permit access to such records. 
Study files may be discarded upon written notification by the Sponsor. To avoid error, the 
Investigator must contact the Sponsor before destroying any records or reports pertaining to 
the study, to ensure that retention is no longer required. Other source documents, such as 
patient’s medical records, must be retained for the maximum time permitted by the hospital 
or institution and until such time when the Investigator is informed by the Sponsor that there 
is no further need to do so. 
In addition, in accordance with the Investigator agreement, the Sponsor should be contacted 
if the site Principal Investigator plans to leave the investigational site so that appropriate 
arrangements can be made. 


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10.14 
Publications 
The main data generated in trial EOCRC1-22 will be published, whether positive or negative, 
that the knowledge generated by performing the trial will become public (including the 
administrative information regarding the trial, including authority/ethic committee 
approvals). Timing of the publication of data will be driven by the Global Coordinating 
Investigator. When based on preliminary data, or on completion of the study, the data 
warrant publication/presentation according to a judgement by the Global Coordinating 
Investigator after collaborative discussions with other involved Investigators (meaning such 
Investigators who have recruited patients to the trial or have had a significant participation 
in the trial by other means), the results may be published/presented, under the auspice of the 
Global Coordinating Investigator, in a recognized scientific journal, or at a scientific 
conference, subject to the provisions of the following process: 
 No publication based on the results obtained at an individual trial site (or group of 
sites) shall be made before the first multicenter publication or presentation unless 
otherwise agreed in writing. Notwithstanding the foregoing, if a multicenter 
publication is not published within twelve (12) months after completion of the 
clinical trial and final lock of the clinical trial database at all research sites that are 
part of the multicenter clinical trial, or any earlier termination or abandonment of the 
clinical trial, or if the Sponsor informs the Principal Investigator that such multicenter 
publication will not take place, or if publication has been agreed otherwise, the site 
Principal Investigator shall have the right to publish or present the methods and 
results obtained at his/her site of the clinical trial.  
 In case a publication/presentation is proposed, the Global Coordinating Investigator 
shall discuss the proposed publication/presentation with the Sponsor and give the 
Sponsor a relevant time (depending on the planned publication situation; e.g. referred 
journal, or scientific conference abstract, submission) to support with data collection 
and analyses which could be the basis for a publication. 
 The Sponsor should have adequate time for review of proposed abstracts, 
manuscripts, and/or other presentation materials before submissions (time is 
dependent on the planned publication situation; adequate time for review before 
submission of a complete manuscripts to a referred journal can be considered as 30 
days, to respond with any requested revisions, including the deletion of confidential 
information which must be done). 
 
 
 
 
 
 


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11 
LITERATURE 
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108. Kim ST, Sohn I, DO IG, et al. Transcriptome analysis of CD133-positive stem cells 
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2077. 


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12 
APPENDICES 
 
12.1 Appendix 1: ECOG performance status 
The performance status grading as outlined below was developed by the Eastern Cooperative 
Oncology Group (ECOG) [101]. 
GRADE  
ECOG PERFORMANCE STATUS 
0 
 Fully active, able to carry on all pre-disease performance without restriction 
1 
 Restricted in physically strenuous activity but ambulatory and able to carry out 
work of a light or sedentary nature, e.g., light house work, office work 
2 
 Ambulatory and capable of all selfcare but unable to carry out any work 
activities; up and about more than 50% of waking hours 
3 
 Capable of only limited selfcare; confined to bed or chair more than 50% of 
waking hours 
4 
 Completely disabled; cannot carry on any selfcare; totally confined to bed or 
chair 
5 
 Dead 
 
 
 


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12.2 Appendix 2: New York Heart Association functional classification  
The New York Heart Association (NYHA) functional classification is described below 
[102]. 
Class 
Patient Symptoms 
Class I (None) 
No limitation of physical activity. Ordinary physical activity does not cause 
undue fatigue, palpitation, or dyspnea (shortness of breath). 
Class II (Mild) 
Slight limitation of physical activity. Comfortable at rest, but ordinary physical 
activity results in fatigue, palpitation, or dyspnea. 
Class III (Moderate) 
Marked limitation of physical activity. Comfortable at rest, but less than 
ordinary activity causes fatigue, palpitation, or dyspnea. 
Class IV (Severe) 
Unable to carry out any physical activity without discomfort. Symptoms of 
cardiac insufficiency at rest. If any physical activity is undertaken, discomfort 
is increased. 
 
 
 
 


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12.3 Appendix 3: National Institute of Health (NIH) deviation definitions 
Notes: 
 the term "subject" has been switched to "patient" by the Sponsor in the below text, 
and 
 in trial EOCRC1-22 the terminology only includes the word "Deviation", which is 
subdivided into "Major Deviations" (corresponding to the "Protocol Violation" term 
as defined by NIH below, when the definition for "Minor Deviation" is not fulfilled), 
and "Minor Deviations" (corresponding to "Minor Protocol Deviation" as defined 
by NIH below). 
NIH Institutional Review Board Professional Administrators Committee Version 5.1  
Regulatory Process Workgroup 11/18/2005 
[http://www.genome.gov/Pages/Research/Intramural/IRB/Deviation_Violation_examples8
-07.pdf; accessed March 13, 2022] [103] 
Protocol Deviations and Violations  
Protocol Deviation - A protocol deviation is any change, divergence, or departure from the 
study design or procedures of a research protocol that is under the Investigator’s control and 
that has not been approved by the EC. Upon discovery, the Principal Investigator is 
responsible for reporting protocol deviations to the EC using the standard reporting form.  
Any change, divergence, or departure from the study design or procedures of a research 
protocol that affects the patient's rights, safety, or well-being and/or the completeness, 
accuracy and reliability of the study data constitutes a protocol violation.  
Changes or alterations in the conduct of the trial which do not have a major impact on the 
patient's rights, safety or well-being, or the completeness, accuracy, and reliability of the 
study data are considered minor protocol deviations.  
Protocol Violation - A protocol violation is a deviation from the EC approved protocol that 
may affect the patient's rights, safety, or well-being and/or the completeness, accuracy and 
reliability of the study data.  
If the deviation meets any of the following criteria, it is considered a protocol violation. 
Example list is not exhaustive.  
I. 
The deviation has harmed or posed a significant or substantive risk of harm to 
the patient.  
Examples:  
 a patient received the wrong treatment or incorrect dose, 
 a patient met withdrawal criteria during the study but was not withdrawn, and 
 a patient received an excluded concomitant medication. 
II. 
The deviation compromises the scientific integrity of the data collected for the 
study.  
Examples:  
 a patient was enrolled but does not meet the protocol's eligibility criteria, 
 failure to treat patient per protocol procedures that specifically relate to primary 
efficacy outcomes (if it involves patient safety it meets the first category above),  
 changing the protocol without prior EC approval, and 
 inadvertent loss of samples or data. 


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III. 
The deviation is a willful or knowing breach of human patient protection 
regulations, policies, or procedures on the part of the Investigator(s).  
Examples:  
 failure to obtain informed consent before initiation of study-related procedures,  
 falsifying research or medical records, and 
 performing tests or procedures beyond the individual's professional scope or 
privilege status (credentialing).  
IV. 
The deviation involves a serious or continuing noncompliance with federal, 
state, local, or institutional human patient protection regulations, policies, or 
procedures.  
Examples:  
 working under an expired professional license or certification,  
 failure to follow federal and/or local regulations, and intramural research or 
clinical center policies, and 
 repeated minor deviations. 
V. 
The deviation is inconsistent with the NIH Human Research Protection 
Program’s research, medical, and ethical principles.  
Examples:  
 a breach of confidentiality, and 
 inadequate or improper informed consent procedure. 
Minor Protocol Deviation is any change, divergence, or departure from the study design or 
procedures of a research protocol that has not been approved by the EC and which DOES 
NOT have a major impact on the patient's rights, safety or well-being, or the completeness, 
accuracy and reliability of the study data.  
 
 
 


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12.4 Appendix 4: Administration-related reactions  
The NCI-CTCAE distinguishes between hypersensitivity reactions and acute infusion 
reactions induced by cytokine release. Despite the different possible mechanisms underlying 
hypersensitivity and infusion reactions, the clinical signs and symptoms associated with 
these reactions overlap.  
Immediate systemic reactions, infusion-related reactions and hypersensitivity reactions, at 
administration of nivolumab can be considered to be common [90, 91]. Systemic reaction at 
administration of EO2040 cannot be excluded either, even if not reported in the early 
development of the similar compound EO2401(see Section 1.3.2).  
NOTE: At administration of the treatment compounds of this trial, immediate treatment of 
severe allergic reactions should be available, including staff well trained in resuscitation, 
IV access for administration of fluids, antihistamines, corticosteroids, and epinephrine 
(adrenaline). 
Treatment of administration-related reactions  
If such reactions would occur, it might manifest with fever, chills, rigors, headache, rash, 
pruritus, arthralgias, hypotension, hypertension, bronchospasm, or other allergic-like 
reactions. All Grade ≥ 3 infusion reactions should be reported within 24 hours to the Sponsor 
via the Medical Monitor and be reported as an SAE if it meets the criteria. Infusion reactions 
should be graded according to NCI-CTCAE v5.0. 
Treatment recommendations are provided below and may be modified based on local 
treatment standards and guidelines, as appropriate: 
 For Grade 1 symptoms: (mild reaction; infusion interruption not indicated; 
intervention not indicated): 
o Remain at bedside and monitor patient until recovery from symptoms. The 
following prophylactic pre-medications are recommended for future 
infusions; diphenhydramine 50 mg (or equivalent), and/or acetaminophen 
325 to 1000 mg orally at least 30 minutes before additional administrations. 
 For Grade 2 symptoms: (moderate reaction requires therapy or infusion interruption 
but responds promptly to symptomatic treatment [e.g. antihistamines, nonsteroidal 
anti-inflammatory drugs, narcotics, corticosteroids, bronchodilators, IV fluids]; 
prophylactic medications indicated for ≤ 24 hours): 
o Stop the infusion, begin an IV infusion of normal saline, and treat the patient 
with diphenhydramine 50 mg IV (or equivalent), and/or acetaminophen 325 
to 1000 mg PO; remain at bedside and monitor patient until resolution of 
symptoms. Corticosteroid and/or bronchodilator therapy may also be 
administered as appropriate. If the infusion is interrupted, then restart the 
infusion at 50% of the original infusion rate when symptoms resolve; if no 
further complications ensue after 30 minutes, the rate may be increased to 
100% of the original infusion rate. Monitor patient closely. If symptoms 
recur, then no further drug will be administered at that visit. 
o For future infusions, the following prophylactic pre-medications are 
recommended: 
diphenhydramine 
50 
mg 
(or 
equivalent), 
and/or 
acetaminophen/paracetamol 325 to 1000 mg PO administered at least 30 
minutes before infusions. If necessary, corticosteroids (up to 25 mg IV of 
hydrocortisone or equivalent) may be used. 


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 For Grade 3 or 4 symptoms: (severe reaction, Grade 3: prolonged [i.e. not rapidly 
responsive to symptomatic medication and/or brief interruption of infusion]; 
recurrence of symptoms following initial improvement; hospitalization indicated for 
other clinical sequelae [e.g. renal impairment, pulmonary infiltrates]. Grade 4: life-
threatening; pressor or ventilatory support indicated): 
o Immediately discontinue infusion of the compound. Begin an IV infusion of 
normal saline and treat the patient as follows: recommend bronchodilators, 
epinephrine 0.2 to 1 mg of a 1:1000 solution for subcutaneous administration 
or 0.1 to 0.25 mg of a 1:10 000 solution injected slowly for IV administration, 
and/or diphenhydramine 50 mg IV with methylprednisolone 100 mg IV (or 
equivalent), as needed. The patient should be monitored until the Investigator 
is comfortable that the symptoms will not recur. Investigators should follow 
their institutional guidelines for the treatment of anaphylaxis. Remain at 
bedside and monitor patient until recovery of the symptoms. Further 
treatment of the patient should be held, and assessments made per the general 
and specific safety rules of the trial. 
Late-occurring hypersensitivity symptoms 
In case of late-occurring hypersensitivity symptoms (e.g. appearance of a localized or 
generalized pruritus within 1 week after treatment), symptomatic treatment may be given 
(e.g. oral antihistamine or corticosteroids). 
 
 
 
 


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12.5 Appendix 5: Management algorithms regarding nivolumab for studies under 
CTCAE version 5.0   
These general guidelines are standard nivolumab protocol safety algorithms (28-Sep-
2020) and recommended for inclusion in any nivolumab containing protocol by Bristol-
Myers Squibb (marketing authorization holder for nivolumab). 
These general guidelines constitute guidance to the Investigator and may be supplemented 
by discussions with the Medical Monitor representing the Sponsor. The guidance applies to 
all immuno-oncology agents and regimens. 
A general principle is that differential diagnoses should be diligently evaluated according to 
standard medical practice. Non-inflammatory etiologies should be considered and 
appropriately treated. 
Corticosteroids are a primary therapy for immuno-oncology drug-related adverse events. 
The oral equivalent of the recommended IV doses may be considered for ambulatory patients 
with low-grade toxicity. The lower bioavailability of oral corticosteroids should be taken 
into account when switching to the equivalent dose of oral corticosteroids. 
Consultation with a medical or surgical specialist, especially prior to an invasive diagnostic 
or procedure, is recommended. 
The frequency and severity of the related adverse events covered by these algorithms will 
depend on the immuno-oncology agent or regimen being used.


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NOTE, below guidance is for nivolumab given as monotherapy! EO2401 administered with the adjuvant Montanide will lead to local 
administration site reaction in approximately 35-50% of patients, with a median duration of 6 weeks (see Section 1.3.6.1). 
DO NOT FOLLOW THE BELOW GUIDANCE FOR ADMINISTRATION SITE REACTIONS RELATED TO EO2040; CONTACT THE 
MEDICAL MONITOR IN ANY CASE OF DOUBT OF HOW SUCH REACTIONS SHOULD BE HANDLED! 
 


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