A Phase II Study of Perioperative QBECO Site Specific Immunomodulator (Qu Biologics®) in Patients With Metastatic Colorectal Adenocarcinoma Within the Liver Undergoing Resection
Phase: PHASE2
Conditions: Colorectal Cancer, Liver Metastases
Sponsor: Sunnybrook Health Sciences Centre

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Protocol Number: 4023 
Version: 4.0  dated 19.AUG.2024 
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PERIOP-06 
A Phase II Study of Perioperative QBECO Site Specific Immunomodulator (Qu Biologics®) in Patients 
with Metastatic Colorectal Adenocarcinoma within the Liver Undergoing Resection 
 
Protocol Number: 4023 
 
 
 
   
Principal Investigators: Dr. Rebecca Auer 
Dr. Paul Karanicolas 
Regulatory Sponsor: Qu Biologics 
Funding Agency: Qu Biologics 
Investigational Product: QBECO Site Specific Immunomodulator (SSI) 
Coordinating Centre: 
 
 
 
 
 
 
 
Centre for Clinical Trial Support (CCTS) 
Sunnybrook Research Institute 
C8-2075 Bayview Avenue 
Toronto, ON M4N 3M5 
Canada 
Email: PERIOP-06@sunnybrook.ca 
 
 
 
 
 
 

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Protocol Number: 4023 
Version: 4.0  dated 19.AUG.2024 
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SPONSOR STATEMENT OF COMPLIANCE 
This study will comply with the International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human use Good Clinical Practice E6 (ICH-GCP), World Medical Association 
Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Participants, as well as 
applicable regulatory and institutional requirements.  
 
Personnel listed below are authorized to sign the protocol and any subsequent protocol amendments on 
behalf of the sponsor:  
Name: 
(Print) 
 
Title:  
(Print) 
 
Signature: 
 
Date of Approval: 
(yyyy-mmm-dd) 
 
 

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PROTOCOL SIGNATURE PAGE 
 
I have read this protocol in its entirety and its appendices. I agree to comply with the requirements of 
the study protocol and procedures for data recording/reporting and acknowledge my responsibility for 
the well-being of each research participant, and to ensure that all persons involved in study activities are 
adequately informed about the protocol, the investigational product, and their trial-related duties. The 
signature below constitutes the agreement to conduct this study in accordance with the REB approved 
protocol, GCP, and applicable regulatory requirements, including confidentiality, ethical guidelines and 
regulations regarding the conduct of research in humans. 
 
Qualified Investigator: 
Name: 
(Print) 
 
Title & Institution:  
(Print) 
 
Signature: 
 
Date of signature: 
(yyyy-mmm-dd) 
 
 

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TABLE OF CONTENTS 
PAGE 
SPONSOR STATEMENT OF COMPLIANCE ...................................................................................................... II 
PROTOCOL SIGNATURE PAGE ...................................................................................................................... III 
TABLE OF CONTENTS .................................................................................................................................... IV 
LIST OF ABBREVIATIONS ............................................................................................................................. VII 
PROTOCOL SUMMARY ................................................................................................................................. IX 
1 
KEY ROLES AND CONTACT INFORMATION ........................................................................... 1 
2 
INTRODUCTION .............................................................................................................................. 3 
2.1 
Background ............................................................................................................................ 3 
2.2 
Site Specific Immunomodulators ........................................................................................... 4 
2.2.1 
SSI Preclinical Data .................................................................................................... 4 
2.2.2 
SSI Clinical Data ......................................................................................................... 4 
2.3 
Rationale ................................................................................................................................ 6 
3 
STUDY OBJECTIVES ..................................................................................................................... 8 
3.1 
Primary Objective ................................................................................................................... 8 
3.2 
Secondary Objectives ............................................................................................................. 8 
3.3 
Correlative Objectives ............................................................................................................ 8 
4 
STUDY DESIGN ............................................................................................................................... 9 
4.1 
General Design ....................................................................................................................... 9 
4.2 
Primary Outcome/Endpoint ................................................................................................... 9 
4.3 
Secondary Outcomes/Endpoints ........................................................................................... 9 
4.4 
Correlative Outcomes .......................................................................................................... 11 
5 
DEFINING AND MEASURING PFS ............................................................................................ 12 
5.1 
Definitions ............................................................................................................................ 12 
5.2 
Measuring PFS ...................................................................................................................... 12 
6 
PARTICIPANT SELECTION AND WITHDRAWAL ................................................................... 13 
6.1 
Inclusion Criteria .................................................................................................................. 13 
6.2 
Exclusion Criteria .................................................................................................................. 13 
6.3 
Participant Recruitment ....................................................................................................... 14 
6.3.1 
Randomization Procedures ..................................................................................... 14 
6.3.2 
Blinding and Unblinding Procedures ....................................................................... 14 
6.4 
Discontinuation of IP and Participant Withdrawal .............................................................. 14 
6.4.1 
Discontinuation of IP and Study Procedures .......................................................... 14 
6.4.2 
Withdrawal .............................................................................................................. 15 
7 
INTERVENTIONS .......................................................................................................................... 17 
7.1 
Investigational Product ........................................................................................................ 17 
7.1.1 
Acquisition, Formulation and Packaging ................................................................. 17 
7.1.2 
Treatment Assignment Procedures ........................................................................ 17 
7.1.3 
Dosage and Administration ..................................................................................... 17 

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7.1.4 
Procedures for Intervention Training ...................................................................... 18 
7.1.5 
Dose Modification ................................................................................................... 18 
7.1.6 
Prior and Concomitant Medications/Treatments ................................................... 19 
7.1.7 
Assessment of Participant Compliance ................................................................... 19 
7.1.8 
Receiving, Storage, Dispensing and Return ............................................................ 19 
8 
STUDY SCHEDULE AND PROCEDURES ................................................................................ 21 
8.1 
Prior to Surgery .................................................................................................................... 23 
8.2 
Day of Surgery ...................................................................................................................... 23 
8.3 
After Surgery ........................................................................................................................ 23 
8.4 
Pregnancy Testing ................................................................................................................ 24 
8.5 
Pregnancy Visit and Follow-Up ............................................................................................ 24 
8.6 
Early Termination Visit ......................................................................................................... 25 
8.7 
Final Study Visit .................................................................................................................... 25 
8.8 
Protocol Deviations .............................................................................................................. 25 
8.9 
Handling Procedures for Blood and Tissue Derived Outcome Measures ............................ 25 
9 
ASSESSMENT OF SAFETY ........................................................................................................ 26 
9.1 
Definitions ............................................................................................................................ 26 
9.1.1 
Adverse Events ........................................................................................................ 26 
9.1.2 
Serious Adverse Events ........................................................................................... 26 
9.1.3 
Unexpected Adverse Event ..................................................................................... 27 
9.1.4 
Adverse Drug Reaction and Unexpected Adverse Drug Reaction .......................... 27 
9.2 
Assessment of an Adverse Event ......................................................................................... 27 
9.2.1 
Relationship (Causality/Relatedness) ..................................................................... 27 
9.2.2 
Expectedness........................................................................................................... 27 
9.2.3 
Seriousness ............................................................................................................. 27 
9.2.4 
Severity.................................................................................................................... 28 
9.3 
Adverse Event Recording ..................................................................................................... 28 
9.4 
Reporting of SAEs and Unanticipated Events ...................................................................... 28 
9.4.1 
Investigator reporting: Notifying the Research Ethics Board ................................. 28 
9.4.2 
Investigator reporting: Notifying the Coordinating Centre .................................... 28 
9.4.3 
Coordinating Centre Reporting of SUADRs: Notifying Health Canada .................... 29 
9.4.4 
Coordinating Centre Reporting of SUADRs: Notifying Sites .................................... 29 
9.4.5 
Coordinating Centre Reporting of SAEs and SUADRs: Notifying the Sponsor ........ 29 
9.5 
Duration of Follow-up for Adverse Events ........................................................................... 29 
9.6 
Pregnancy and Adverse Events ............................................................................................ 29 
9.7 
Data and Safety Monitoring Board ...................................................................................... 30 
10 
SITE MONITORING, AUDITING, AND INSPECTING ............................................................. 31 
10.1 
Site Monitoring Plan............................................................................................................. 31 
10.2 
Auditing and Inspecting ....................................................................................................... 31 
11 
STATISTICAL CONSIDERATIONS ............................................................................................ 32 
11.1 
Study Hypotheses ................................................................................................................. 32 

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11.2 
Sample Size Considerations ................................................................................................. 32 
11.3 
Planned Safety Analyses ...................................................................................................... 32 
11.4 
Stopping Rules ...................................................................................................................... 32 
11.5 
Final Analysis Plan ................................................................................................................ 33 
12 
DATA HANDLING AND RECORD KEEPING ............................................................................ 35 
12.1 
Confidentiality ...................................................................................................................... 35 
12.2 
Source Documents ............................................................................................................... 35 
12.3 
Data Management Responsibilities ..................................................................................... 36 
12.4 
Data Capture and Case Report Forms .................................................................................. 36 
12.5 
Records Retention ................................................................................................................ 36 
12.6 
Clinical Trial Registration ...................................................................................................... 36 
13 
QUALITY CONTROL AND QUALITY ASSURANCE ............................................................... 37 
14 
ETHICS CONSIDERATIONS ....................................................................................................... 38 
14.1 
Ethical Standard ................................................................................................................... 38 
14.2 
Research Ethics Board .......................................................................................................... 38 
14.3 
Consent ................................................................................................................................ 38 
15 
PUBLICATION/DATA SHARING POLICY ................................................................................. 39 
16 
REFERENCES ............................................................................................................................... 40 
APPENDIX A: CIOMS FORM ......................................................................................................................... 44 
APPENDIX B: CONTRACEPTIVE GUIDANCE ................................................................................................. 45 
 
 

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LIST OF ABBREVIATIONS 
The following abbreviations describe terms, documents, and study personnel used in the conduct of this 
study protocol. 
ADR 
Adverse Drug Reaction 
AE 
Adverse Event/Adverse Experience 
CBC 
Complete Blood Count 
CEA 
Carcinoembryonic Antigen 
CIOMS 
Council for international Organizations of Medical Sciences 
CTCAE 
Common Terminology Criteria for Adverse Events 
CCTS 
Centre for Clinical Trial Support 
CRF 
Case Report Form 
CRLM 
Colorectal Liver Metastases 
CT 
Computed Tomography  
ctDNA 
Circulating Tumour DNA 
DSMB 
Data and Safety Monitoring Board 
DVT 
Deep Vein Thrombosis 
FFPE 
Formalin-fixed paraffin-embedded 
GI 
Gastrointestinal 
H&E 
Hematoxylin and Eosin 
HIV 
Human Immunodeficiency Virus  
IB 
Investigator’s Brochure 
IBD 
Inflammatory Bowel Disease 
ICH 
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
ICU 
Intensive Care Unit 
IP 
Investigational Product 
ICF 
Informed Consent Form 
IV 
Intravenous 
GCP 
Good Clinical Practice 
LSIR 
Local Skin Immune Response 
MDSC 
Myeloid-Derived Suppressor Cell 
MedDRA 
Medical Dictionary for Regulatory Activities 
MODS 
Multiple Organ Dysfunction Syndrome 
NK 
Natural Killer 

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OS 
Overall Survival 
PFS 
Progression-Free Survival 
PHI 
Personal Health Information 
PHIPA 
Personal Health Information Protection Act 
PI 
Principal Investigator 
POD 
Postoperative Day 
QoR 
Quality of Recovery 
REB 
Research Ethics Board 
RCT 
Randomized Controlled Trial 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SC 
Subcutaneous 
SOP 
Standard Operating Procedure 
SSI 
Site Specific Immunomodulator 
SUADR 
Serious and Unexpected Adverse Drug Reaction 
TME 
Tumour Microenvironment 
VTE 
Venous Thromboembolism 
WOCBP 
Women of Childbearing Potential 
 

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PROTOCOL SUMMARY 
Protocol Title  
A Phase II Study of Perioperative QBECO Site Specific Immunomodulator 
(Qu Biologics®) in Patients Undergoing Resection of Metastatic Colorectal 
Adenocarcinoma within the Liver 
Protocol Number 
4023 
Phase 
II 
Study Design 
Multicentre, blinded, randomized, placebo-controlled trial  
Study Duration 
7 years 
Setting 
Tertiary referral centres in Canada 
Sample Size 
115 participants 
Main Inclusion Criteria 
Adult patients planned to undergo resection of colorectal liver metastases 
for complete clearance of all visible disease 
Primary Outcome: 
Progression-free survival  
Secondary and 
Correlative Outcomes: 
ctDNA, side-effect profile, quality of recovery, 5-year overall survival, and 
blood-based measurements of postoperative immune dysfunction  
Investigational Product 
and Planned Use 
QBECO is a site specific immunomodulator (SSI) designed to promote 
innate immune responses in the gastrointestinal tract and related organs, 
including the liver.  
QBECO or placebo will be administered according to the following 
regimen: 0.1mL SC q2days for 11-120 days preoperatively, and 41 days 
postoperatively. 
Statistical Analysis: 
Descriptive statistics, time-to-event analysis, and regression modelling 
 
 
 

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1 KEY ROLES AND CONTACT INFORMATION 
Principal Investigators:   
Dr. Paul Karanicolas, MD, PhD, FRCSC, FACS 
Sunnybrook Health Sciences Centre 
2075 Bayview Avenue, Room T2-16 
Toronto, ON M4N 3M5 
Phone: 416-480-4774 
Fax: 416-480-6002 
Email: paul.karanicolas@sunnybrook.ca 
 
 
Dr. Rebecca Auer, MD, MSc, FRCSC 
The Ottawa Hospital, General Campus 
501 Smyth Road, CCW 1617 
Ottawa, ON K1H 8L6 
Email: rauer@toh.ca 
 
Regulatory Sponsor: 
Qu Biologics 
4475 Wayburne Drive, Suite 305 
Burnaby, BC V5G 4X4 
 
Academic Research Organization: 
Centre for Clinical Trial Support (CCTS) 
Sunnybrook Research Institute 
C8-2075 Bayview Avenue 
Toronto, ON M4N 3M5 
 
Primary Funding Agency: 
Qu Biologics 
4475 Wayburne Drive, Suite 305 
Burnaby, BC V5G 4X4 
 
Additional Supporting Agencies: 
Foundation Medicine, Inc. 
150 Second Street 
Cambridge, MA 02141 
USA 
 
Personal Genome Diagnostics Inc  
3600 Boston St., Suite 10 
Baltimore, MD 21224 
 
NKMAX 
1172 Dolma-ro 
Bundang-gu, Seongnam-si, Gyeonggi-do 13605 
Republic of Korea  
 
 
 

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Clinical Laboratory: 
Dr. Rebecca Auer 
The Ottawa Hospital, General Campus 
501 Smyth Road, CCW 1617 
Ottawa, ON K1H 8L6 
 

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2 INTRODUCTION  
This study document is a protocol for research involving human participants. This study is to be conducted 
according to Canadian and international standards, and in compliance with the protocol, the International 
Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good 
Clinical Practice E6 (GCP), World Medical Association Declaration of Helsinki: Ethical Principles for Medical 
Research Involving Human Participants, as well as applicable regulatory and institutional requirements 
and research policies.  
2.1 
Background 
Surgical trauma induces a profound suppression of immune function that occurs within hours, lasts for 
days to weeks, and is directly correlated with the severity of the surgical procedure.1 Postoperative 
immune suppression has serious implications in cancer, where it has been shown to contribute to cancer 
regrowth and metastases.2–6 The significance of surgery-induced immune suppression in patients with 
cancer presents a critical target for future therapies. 
The mechanism of postoperative immune suppression is largely mediated by natural killer (NK) cell 
dysfunction. NK cells are innate lymphocytes that play a pivotal role in immune surveillance and clearance 
of metastases. In response to surgical trauma, the bone marrow is stimulated to rapidly release myeloid 
cells into the blood.7,8 This “emergency myelopoiesis” has evolved to protect the host, however, some of 
these myeloid cells have immunosuppressive properties. Studies from Dr. Auer’s lab have demonstrated 
that 
these 
myeloid-derived 
suppressor 
cells 
(MDSCs) 
are 
responsible 
for 
postoperative 
immunosuppression, including NK cell dysfunction, and the resulting proliferation of cancer metastases.3  
In addition to surgical trauma, cancers themselves induce NK cell dysfunction, which is significant and 
measurable across cancer types.3,4,9 However, Dr. Auer’s lab has shown that the magnitude of cancer-
induced NK cell dysfunction pales in comparison to NK cell dysfunction following surgical stress.9 Most 
striking is that once suppressed, no amount of stimulation in in vitro NK cell assays can recover the defect 
and restore NK cell effector functions.2,3 However, preoperative hyperactivation, while unable to reverse 
the effects of surgery, can attenuate them.2,3,9,10 
Perioperative trained immunity is hypothesized to prevent or attenuate the effects of postoperative 
immune suppression. Trained immunity is a process whereby exposure to microbial stimuli induces long 
lasting epigenetic and functional changes in innate immune cells such as NK cells. These changes occur 
predominantly in the bone marrow, where microbial modulation of myelopoiesis results in the production 
of epigenetically modified macrophages better able to address the next immunological threat, whether 
that be another pathogen or cancer.11,12 Following immunological training, both myeloid and NK cells have 
enhanced cytokine production in response to stimuli with associated improved cytotoxicity.13 Given the 
postoperative period is associated with the release of MDSCs leading to NK cell dysfunction, preoperative 
innate immune training may prevent or attenuate the negative immune sequelae of surgery. However, 
there are no available cancer treatments directed at innate immune training, nor have any available 
therapies proven effective in alleviating postoperative immune suppression. 

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2.2 
Site Specific Immunomodulators 
Qu Biologics is a Canadian biotechnology company that has developed a novel class of 
immunomodulators, called Site Specific Immunomodulators (SSIs). They are designed, in the context of 
cancer, to induce trained immunity in organs at risk for micrometastases. The anti-cancer effects of SSIs 
are mediated through a number of mechanisms. SSIs relieve the immune suppression that dominates the 
tumour microenvironment (TME), make cancer cells more visible to the immune system through 
upregulation of stress-induced molecules such as NKG2D ligands, recruit M1 macrophages into the TME, 
down-regulate inhibitory molecules such as PD-1 and PD-L1, and enhance NK cell cytotoxicity.14 
QBECO is an SSI formulated from inactivated E. coli bacteria that is specifically designed to target 
pathologies of the gastrointestinal (GI) tract and related organs, such as the liver. This trial will test the 
hypothesis that in patients undergoing resection of colorectal liver metastases (CRLMs), perioperative 
treatment with QBECO will attenuate the postoperative immune suppression and will improve 
progression-free survival (PFS).  
2.2.1 
SSI Preclinical Data 
Preclinical studies found that perioperative administration of an SSI designed to target lung pathologies 
(QBKPN) attenuated pulmonary metastases and improved NK cell function in the postoperative period 
(unpublished data from Dr. Auer’s lab). The efficacy of QBKPN was not limited to lung cancer cell lines and 
demonstrated reduced tumour burden in models of lung metastases for melanoma. These unpublished 
data from Dr. Auer’s lab provided proof-of-concept for the use of SSIs in cancer. 
With regards to QBECO, preclinical studies have demonstrated reduced formation of colorectal cancer 
and prolonged survival in murine models of metastatic disease.15 The immune response following 
administration of QBECO was directed to the GI and peritoneal regions, corresponding to the endogenous 
niche for E. coli. In a murine model of metastatic colorectal cancer (MC38 in C57BL/6 mice), perioperative 
QBECO resulted in a significant reduction in hepatic metastases (unpublished data, Dr. Auer’s lab).  
In terms of preclinical safety of QBECO, no adverse events (AEs) or notable toxicities in mice were 
observed after 28 days of therapy with 1,168 times the dose, by body weight, to be used in this study. 
Further details regarding these safety data are provided in the Investigator’s Brochure (IB). 
2.2.2 
SSI Clinical Data 
QBECO SSI is an inactivated whole-cell bacterial vaccine. Inactivated whole-cell bacterial vaccines have 
been in use for almost 150 years, including ongoing use of whole-cell inactivated B. pertussis vaccine in 
millions of infants annually. The side effect profile of this class of drugs is well characterized and 
understood, including: 
 
Risk of anaphylaxis: Rare; particularly rare with inactivated whole-cell bacterial vaccines like 
QBECO that have no excipients. 
 
Exotoxin related risk: Washing steps (x3) in QBECO manufacturing process eliminates this risk. 
 
Endotoxin systemic immune activation (e.g., pyrexia) due to high free endotoxin levels: The 
QBECO manufacturing process results in levels of free endotoxin in QBECO dosing that is orders 
of magnitude below levels that induce pyrexia/systemic immune activation, as described below. 

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For example, B. pertussis vaccine, administered to infants, has an average free endotoxin dose of 
approximately 60,000 Endotoxin Units (EU)/ml and is administered as a 5 mL dose = 300,000 EU per dose. 
The QBECO clinical lot has a free endotoxin dose of approximately 3,350 EU/mL and is administered as a 
0.1 mL dose = 335 EU per dose, which is ≈ 900 times lower. Providing further margins of safety, (1) risk of 
endotoxin systemic immune activation is based on EU/Kg body weight and thus, a 335 EU dose in adults 
is far safer than a 300,000 EU dose in infants, and (2) QBECO is administered subcutaneously, which is 
significantly less likely to cause endotoxin systemic immune activation than intramuscular injection, which 
is the method of administration of the B. pertussis vaccine.  
QBECO manufacture and formulation is based on other inactivated whole cell bacterial products that are 
or were commercially available for more than 40 years, such as Mixed Respiratory Vaccine (Bayer 
Corporation, Hollister-Stier Laboratories, and Stallergenes) and Polyvaccinum Forte (Biomed, Poland). As 
with QBECO SSI, these inactivated whole-cell bacterial vaccines were manufactured by cultivation, 
inactivation, washing with saline, and resuspension in saline, with a similar concentration, dose (up to 0.4 
mL, which is 4x higher than the PERIOP-06 QBECO dose), dose frequency, and treatment duration, and 
were used for the treatment of relatively benign conditions such as rhinitis, sinusitis, bronchitis, and 
cystitis, providing historic precedent of safety. The free endotoxin level of one of these commercially used 
products, Polyvaccinum Forte, which contains inactivated whole-cell E. coli in addition to other inactivated 
bacterial species, was tested, using the endotoxin assay (Endosafe [Charles River]) used in QBECO release 
and stability testing, and was found to be 24,000 EU/mL, which is ≈ 7x higher than the free endotoxin level 
of PERIOP-06 QBECO clinical lot (i.e., 3,350 EU/mL), thus, affording QBECO clinical lot additional significant 
margins of safety.  
The half-life of free endotoxin in humans is 4-5 minutes with 100% cleared after 30 minutes and thus, 
there is no cumulative effect on the level of free endotoxin with the every second day dosing schedule 
used in this study.16 
Over 360 patients have been treated with SSIs in Phase I/II trials and compassionate use programs. SSIs 
have been well tolerated by patients with >90% compliance rates and favorable side effect profiles to date 
that are similar to those of traditional whole-cell inactivated vaccines.  
QBKPN SSI has been administered to a total of 111 patients with advanced lung cancer or lung metastases, 
including 105 patients in a compassionate use program and 6 patients in an open-label, single-arm Phase 
1/2 clinical study of 6 adults with recurrent lung adenocarcinoma.14 A retrospective case-matched analysis 
(n = 7 matched cases) of patients with advanced lung adenocarcinoma in the compassionate use program 
found a 14-month median survival advantage associated with QBKPN treatment. In the Phase 1/2 study 
(n = 6), all patients demonstrated stable tumour burden during the 12 weeks of QBKPN treatment and 
there was a trend towards decreased proportion of circulating immunosuppressive PD-1 and PD-L1 
positive myeloid cells, indicative of immune activation. No serious adverse events (SAEs) attributed to 
QBKPN treatment occurred in the total of 111 participants treated. 
QBECO SSI has been administered to a total of 208 patients, including 121 patients in compassionate use 
programs in oncology (n = 109), Crohn’s disease (n = 10), and ulcerative colitis (n = 2), and 87 patients in 
Phase 1/2 inflammatory bowel disease (IBD) clinical trials in Crohn’s disease (n = 76) and ulcerative colitis 
(n = 11).  

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In the oncology compassionate use program, QBECO SSI was well tolerated with no severe adverse events 
attributed to QBECO treatment in the 109 patients treated. A retrospective case-matched analysis (n = 9 
matched cases) of patients with advanced colorectal adenocarcinoma in the compassionate use program 
found a 6-month median survival advantage associated with QBECO treatment. 
In IBD, it was hypothesized that QBECO treatment would activate a gastrointestinal (GI) tract-specific 
innate immune response, leading to restoration of productive innate immunity, re-establishment of 
barrier function, clearance of bacterial infection, restoration of autophagy, and reduced IBD activity. The 
first IBD trial (n = 11 subjects with moderate-to-severe ulcerative colitis treated with QBEC0 SSI every 
second day for up to 52 weeks) found 8 of 11 subjects (73%) experienced clinical improvement as 
measured by Mayo score (≥ 3 point decrease), 6 of 11 subjects (55%) experienced endoscopic 
improvement, and 6 of 11 subjects (55%) experienced histological improvement.17 The second IBD trial (n 
= 68 subject randomized placebo controlled study in subjects with moderate-to-severe Crohn’s diseases 
treated with QBECO or placebo every second day for up to 16 weeks) found greater improvement in 
Crohn’s disease activity index (CDAI) score in QBECO treated subjects than in subjects treated with 
placebo.18 The third IBD trial (n = 20 subject open-label study in subjects with moderate-to severe Crohn’s 
disease treated for up to 52 weeks) demonstrated evidence of endoscopic and histological response, as 
well as clinical symptom response and maintenance over the 52-week treatment period. In these three 
IBD trials, QBECO was again well tolerated, with the majority of AEs being mild to moderate. Local skin 
immune response at the site of injection and mild transient fatigue were the most commonly reported 
AEs. Safety data from these clinical trials are available in the IB, along with a detailed description of the 
pharmacology, efficacy, and safety of QBECO SSI. 
Collectively, these data support the safety and potential efficacy of SSI treatment to overcome the 
immune suppression that enables cancer growth and metastases.  
2.3 
Rationale 
This multicentre, blinded, phase II, randomized, placebo-controlled trial will evaluate the effect of QBECO 
on PFS in patients undergoing resection of CRLMs. This trial is motivated by the promising preclinical and 
clinical data supporting the safety and efficacy of QBECO in attenuating postoperative 
immunosuppression and the resulting proliferation of cancer.  
Patients undergoing resection of CRLMs were chosen as the study population due to their survival 
characteristics and potential to benefit from the intervention. Approximately 25-30% of patients 
diagnosed with colorectal adenocarcinoma develop liver metastases during the course of their disease.19–
21 Surgery is the only potentially curative treatment for patients with liver metastases, however following 
liver resection, 75% of patients will still experience a recurrence of their cancer.22 Three-year PFS is 
estimated to be approximately 42% in patients undergoing CRLM resection.22 Neoadjuvant and adjuvant 
systemic therapies have demonstrated improvements in PFS for patients with resectable CRLMs, however 
a clear overall survival (OS) benefit has not been shown.22–24 The high incidence of disease progression, 
yet noteworthy potential for cure, make patients undergoing resection of CRLM an ideal population for 
investigation. If this trial demonstrates improved cancer outcomes, SSIs have the potential to be 
transformative in treating all patients undergoing cancer surgery.  
Given the clinical safety data to date in the 208 subjects treated with QBECO, the broad safety margins in 
non-clinical toxicology studies demonstrating no notable toxicity using doses up to 1,168 times (by body 

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weight) the QBECO dose of this study, the preclinical data demonstrating that QBECO treatment reduces 
metastases in a peri-operative colorectal cancer liver metastases model, and the urgent need to improve 
outcomes in stage 4 colorectal cancer patients undergoing resection of liver metastases for which there 
are no current adjuvant treatments that improve overall survival, it is likely that QBECO has a favorable 
risk-benefit profile in this study population. 

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3 STUDY OBJECTIVES 
3.1 
Primary Objective 
The primary objective of this randomized controlled trial (RCT) is to determine if QBECO administered 
perioperatively can improve PFS in adult patients undergoing resection of CRLMs for complete clearance 
of metastatic disease. 
3.2 
Secondary Objectives 
Secondary objectives will be to:  
1. Determine the effect of QBECO on the frequency and kinetics of clearance (and recurrence) of 
circulating tumor DNA (ctDNA) in the postoperative period and further evaluate the use of ctDNA 
as part of ongoing surveillance. 
2. Determine the side-effect profile of perioperative QBECO. This will include an assessment of AEs 
and postoperative complications, and whether the administration of QBECO delays or prevents 
patients from proceeding to surgery. 
3. Determine the effect of QBECO on patient-reported quality of recovery (QoR). 
4. Determine the effect of QBECO on 5-year OS. 
3.3 
Correlative Objectives 
Correlative objectives may be to:  
1. Determine the effect of QBECO on immune phenotype and dysfunction in the perioperative 
period, as measured by phenotypic and functional NK cell and myeloid cell assays, such as 
cytokine secretion, cytotoxicity, and flow cytometry. 
2. Determine the effect of QBECO on immune gene and protein expression in resected hepatic 
metastases and in the blood during the perioperative period using techniques such as 
immunohistochemistry, RNA sequencing, extracellular and intracellular flow cytometry and mass 
spectrometry. 

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4 STUDY DESIGN 
4.1 
General Design 
This will be a multicentre, phase II, blinded, parallel group RCT. Approximately 115 participants will be 
randomized 1:1 to receive QBECO or placebo. Participants will be followed for 5 years after surgery. The 
trial design schematic is presented below: 
Figure 1: Study Schema 
 
4.2 
Primary Outcome/Endpoint 
The primary outcome will be the PFS rate. PFS will be defined as the time from enrollment to the first 
event that is either locoregional recurrence (of the resected primary colorectal adenocarcinoma or the 
resected ± intraoperatively ablated metastatic disease), distant metastases, or death from any cause. 
Section 5 further defines PFS and its measurement. 
4.3 
Secondary Outcomes/Endpoints 
The following list constitutes the secondary outcomes and includes a brief rationale for their 
measurement. 

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1. Clearance of ctDNA. A substantial body of research has demonstrated associations between 
ctDNA and cancer progression.25,26 Timing of measurement will be at baseline (which will occur 
within 28 days prior to the first dose of Investigational Product (IP) preoperatively on the day of 
surgery, and postoperatively. A sample of fresh tumour tissue will be obtained from a 
representative hepatic tumour and from the primary colorectal tumour, if resection occurs at the 
same operation as the liver resection, along with a sample of normal adjacent tissue (one of each 
type per surgical specimen) to tailor the ctDNA assay to the individual participant’s tumour profile. 
If fresh tumour tissue is not available from the resected specimen at the time of the surgery, 
archival tissue will be requested. If there is insufficient material from the fresh and archival tissue 
to undertake the ctDNA assay, tumour tissue from a prior resection or biopsy of the participant’s 
colon cancer will be requested. If disease progression is detected, ctDNA will also be measured at 
that time. 
2. Side-effect profile of QBECO. This will include an evaluation of AEs and postoperative 
complications in the treatment and control groups. AEs will be measured through the procedures 
specified in Section 9. A postoperative complication will be defined as any deviation from the 
normal postoperative course.27 All clearly related signs, symptoms, and abnormal diagnostic 
procedures should be recorded in the source document and should be grouped under one 
diagnosis. The deviation must be possibly or probably related to the surgery and must occur 
postoperatively. The modified Clavien-Dindo system will be used to define and classify 
postoperative complications.27 Complications of particular interest will be evaluated individually 
for associations with the intervention and include the following: 
 
Surgical site infection. This will be stratified according to the Centers for Disease Control 
and Prevention National Healthcare Safety Network definitions of superficial incisional, 
deep incisional, organ/space surgical site infection.28 
 
Postoperative hemorrhage requiring transfusion, radiologic intervention, or surgical 
intervention. 
 
Bile leak. This will be defined and graded according to the International Study Group of 
Liver Surgery classification.29 
 
Post hepatectomy liver failure. This will also be defined and graded according to the 
International Study Group of Liver Surgery classification.30 
 
Venous thromboembolism (VTE). This will be defined as either a deep vein thrombosis 
(DVT) or pulmonary embolism. DVT may be diagnosed by either ultrasonography or 
contrast venography. Pulmonary embolism may be diagnosed by either a high probability 
V/Q scan, positive pulmonary angiogram, or a CT demonstrating an intraluminal filing 
defect in a pulmonary vessel.  
3. Quality of recovery, as measured by the QoR-40. The QoR-40 is a validated tool designed to 
measure the early postoperative health status of patients.31,32 The tool has been widely used as a 
patient-reported outcome measure of recovery after surgery and has been translated into 
multiple languages.33–36 Timing of measurement will be at baseline (which will occur within 28 
days prior to the first IP dose on postoperative day (POD) 4 (±1), and 6 weeks postoperatively. The 
survey may be administered in person or virtually by telephone or video call.  
4. Five-year overall survival. This will be measured from date of enrollment to the date of death. 

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4.4 
Correlative Outcomes 
The following list constitutes correlative outcomes. These outcomes will be optional depending on 
funding and trial results. 
1. Immune phenotype and dysfunction as measured by functional assays. Dr. Auer’s lab has 
demonstrated significantly impaired NK cell function, including cytokine secretion and cytotoxicity 
in response to surgical stress.9 The laboratory has also demonstrated that innate immune cells, 
including myeloid cells, have a differential cytokine secretion profile following surgery. SSIs have 
been shown to induce a proinflammatory cytokine profile in innate immune cells. We may use 
cytokine release assays to determine the effect of surgery on immune cell cytokine secretion and 
cytotoxicity assays to measure tumour cell killing. Timing of measurement will be at baseline 
(which will occur within 28 days prior to the first IP dose preoperatively on the day of surgery, and 
postoperatively. If disease progression is detected, the outcome may also be measured at that 
time.  
2. Immune gene and protein expression as measured by mRNA expression levels and protein 
levels. Immune cells from the blood and/or the tumour microenvironment may be evaluated 
using RNA sequencing, immunohistochemistry, or mass spectrometry to determine the effect of 
SSI on gene expression at baseline and in response to stimulation. Flow cytometry for intracellular 
and extracellular proteins may be performed on frozen or fixed lymphocytes for immunoprofiling. 
Plasma will also be collected and may also be analyzed. Timing of measurement will be at baseline 
(which will occur within 28 days prior to the first IP dose, preoperatively on the day of surgery, 
and postoperatively. If disease progression is detected, the outcome will also be measured at that 
time.  
3. C-reactive protein (CRP) and serum albumin. There is evidence to suggest that markers of 
systemic inflammation are associated with worse outcome in advanced cancers.37,38 Specifically, 
elevated preoperative CRP and hypoalbuminemia may be prognostic of worse survival after 
resection of CRLM.39 Alternatively, these levels may be increased with SSI administration and be 
associated with a better prognosis and postoperative recovery. Preoperative CRP and Albumin 
may be measured in this trial and assessed for associations with survival and postoperative 
outcomes.  
Participants enrolled at The Ottawa Hospital will be evaluated for the same secondary and correlative 
outcomes as the other study sites with the potential for additional measurement. Participants at The 
Ottawa Hospital will also be evaluated for the additional correlative outcome on CD33+ cells, which can 
only be evaluated when the cells are processed fresh. These assay is described below: 
4. CD33+ myeloid cell suppression. CD33+ myeloid cells contribute to postoperative immune 
suppression and NK cell suppression.3 An NK cell killing assay will be performed to measure CD33+ 
suppression of NK cells, when possible. This will be measured for participants enrolled at the 
Ottawa Hospital. This outcome will not be measured for participants from other centres. Timing 
of measurement will be at baseline (which will occur within 28 days prior to the first IP dose and 
postoperatively. If disease progression is detected, the outcome will also be measured at that 
time.  

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5 DEFINING AND MEASURING PFS 
As PFS is the primary outcome of this study, it is vital that it is precisely defined and documented.  
5.1 
Definitions 
PFS will be defined as the time from enrollment to the first event that is either locoregional recurrence 
(of the resected primary colorectal adenocarcinoma or the resected ± intraoperatively ablated metastatic 
disease), distant metastases, or death from any cause. The identification of a new primary colorectal 
adenocarcinoma or other primary cancer will not be considered an event. Loss to follow-up will be 
censored. This definition of PFS was chosen to align with a consensus agreement on the definition of 
relapse-free survival in the context of adjuvant colorectal cancer studies.40 “Progression” was selected as 
a more appropriate label for events than “relapse” given participants in this trial will have metastatic 
disease at the time of enrollment.  
The following additional occurrences will be considered events at the date of surgery: (1) the identification 
of extrahepatic metastases at the time of the operation that are not resected or (2) if the participant does 
not undergo resection or ablation of all known disease during the operation (unless the rationale for this 
is a complete clinical response). Events will not include the intraoperative identification of previously 
undocumented liver metastases if the metastases are resected or ablated at the time of surgery. 
Distinguishing a new primary colorectal adenocarcinoma from a recurrent primary will be at the discretion 
of the medical care team and investigators.  
5.2 
Measuring PFS 
PFS will be measured from the date of enrollment.  
Disease recurrence is defined as the clinical presence of cancer that has either been confirmed by biopsy, 
has had treatment initiated, or is documented in the treating physician’s notes. The date of disease 
recurrence will be defined as the date of the first suspicious record of the malignant lesion but the date 
cannot be before the date of surgery.  
For participants who do not undergo resection or ablation of all known disease (Including any extrahepatic 
metastases identified intraoperatively, the event date will be the date of surgery (unless the rationale is 
a complete clinical response). 
For participants who, in retrospect, had extra-hepatic disease present before surgery that was not 
recognized, the date of progression will be defined as the date that the lesion in question progressed in 
size or became apparent to the clinical team  

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6 PARTICIPANT SELECTION AND WITHDRAWAL   
We will enroll 115 participants as explained by the sample size calculations in Section 11.2. Participant 
recruitment is anticipated to take approximately 2 years. The subsequent sections describe the inclusion 
and exclusion criteria that must be met at the time of enrollment. Participants will not be excluded on the 
basis of any neoadjuvant chemotherapy or planned adjuvant chemotherapy. 
6.1 
Inclusion Criteria 
Each participant must meet all the following inclusion criteria: 
1. Adults aged 18 years or older at time of enrollment. 
2. Pathologic diagnosis of colorectal carcinoma with clinical diagnosis of liver metastases. 
3. Planned to undergo resection of liver lesions for complete clearance of all visible metastatic 
disease. This will include those who may undergo synchronous resection of the primary colorectal 
cancer and/or those who may receive a combination of surgery and ablation to treat all lesions. 
4. Computed Tomography (CT) of the chest, abdomen, and pelvis with intravenous (IV) contrast 
within 6 weeks prior to enrollment. 
5. MRI of the liver within 6 weeks prior to enrollment OR within 6 weeks prior to starting 
neoadjuvant chemotherapy OR during neoadjuvant chemotherapy (for patients treated with 
chemotherapy).  
6. Planned to receive the last dose of neoadjuvant chemotherapy at least 25 days prior to surgery 
(for patients treated with neoadjuvant chemotherapy).  
7. Agree to comply with the contraceptive requirements of the protocol when applicable (see 
Appendix B). 
8. Willing and able to either perform subcutaneous injections according to the study protocol, or 
receive the injections from a caregiver delegated by the participant. 
9. Able to provide informed consent or has a substitute decision maker capable of providing consent 
on their behalf. 
6.2 
Exclusion Criteria 
All participants meeting any of the following exclusion criteria at baseline will be excluded from 
participation in this study:  
1. Prior or current evidence of extrahepatic metastases. Patients with small (< 1.0 cm) indeterminate 
pulmonary nodules may be included at the investigator’s discretion. 
2. Prior hepatic arterial infusion or embolization. Prior portal vein embolization, ablation, or liver 
resection are permitted. 
3. Patients with any invasive cancer history other than colorectal cancer in the last 5 years. In situ 
disease (e.g., melanoma in situ, ductal carcinoma in situ of the breast) or non-melanoma skin 
cancers are permitted. 
4. Patients with a documented history of clinically severe autoimmune disease or a syndrome that 
requires systemic steroids or immunosuppressive agents. This includes patient requiring systemic 
treatment with either corticosteroids (> 10 mg daily prednisone equivalent, or depot 
corticosteroids in the 6 weeks before enrollment) or immunosuppressant drugs (such as 
azathioprine, tacrolimus, cyclosporine, etc.) within the 14 days prior to enrollment or a reasonable 

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expectation that the patient may require such treatment during the course of the study. Inhaled 
or topical or inter-articular steroids, and adrenal replacement steroid doses ≤ 10 mg daily 
prednisone equivalent, are permitted in the absence of active autoimmune disease. Steroids used 
for premedication prior to chemotherapy or as part of a chemotherapy regimen are allowed. 
5. Patients with known active human immunodeficiency virus (HIV), Hepatitis B, or Hepatitis C 
infections. 
6. Pregnant patients or those who are nursing an infant. 
6.3 
Participant Recruitment  
Participants will be recruited at each of the participating sites. A member of the circle of care will introduce 
the study to potential participants and ask if they are willing to discuss the study with a member of the 
research team. A member of the research team will obtain informed consent. The participant will be 
considered enrolled after providing informed consent.  
Patients must have an anticipated date of surgery of at least 11 days from enrollment, since participants 
with anticipated dates of surgery less than 11 days from enrollment would not be able to receive the 
minimum of 6 preoperative doses of IP if enrolled. 
6.3.1 
Randomization Procedures  
Randomization will occur using a secure web-based system. Participants will be randomized 1:1 in 
permuted blocks to receive either QBECO or placebo according to the dosing schedule outlined in Section 
7.1.3. Randomization will be stratified according to centre and whether the participant received or is 
receiving neoadjuvant chemotherapy in advance of their planned liver resection. 
6.3.2 
Blinding and Unblinding Procedures 
Blinded stakeholders will include participants, clinicians, data collectors, outcome adjudicators, and data 
analysts.41 The study blind may only be broken if the safety of the participant is at risk and the treatment 
plan relies on knowledge of study allocation. If knowledge of the treatment assignment is absolutely 
necessary, the blind may be broken only with the permission of a Principal Investigator. If unblinding 
occurs, the circumstances around the unblinding will be documented. 
6.4 
Discontinuation of IP and Participant Withdrawal 
6.4.1 
Discontinuation of IP and Study Procedures   
The clinical scenarios in which participants will discontinue IP and study procedures are described below 
and summarized in Appendix C.  
At their own discretion, participants may discontinue IP for any reason at any time. These participants will 
continue all study related procedures and visits if they are agreeable..  
Investigators may also discontinue the IP for reasons including, but not limited to, safety or behavioral 
concerns. These patients continue all study related procedures and visits.  
IP should be held postoperatively if a participant has an unplanned admission to an Intensive Care Unit 
(ICU) due to the development of multiple organ dysfunction syndrome (MODS). MODS is defined as the 

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development of a physiologic derangement involving 2 or more organ systems arising as a result of a 
physiologic insult.42,43 Whether a participant meets the definition of MODS will be at the discretion of the 
treating clinicians. Treatment should be resumed when the Qualified/Sub-Investigator feels it is safe to 
do so. Other study related procedures will continue as scheduled  
If a participant does not undergo surgery for any reason other than an apparent clinical complete response 
to neoadjuvant therapy (i.e., progression, the participant is too unwell, participant preference, or >120 
days have elapsed since starting IP), the participant will discontinue IP as soon as possible and be 
monitored for AEs for 30 days after the last dose. PFS and OS will be followed as scheduled. ctDNA will be 
measured once within 14 days of the decision to not undergo surgery. Quality of recovery and correlative 
outcomes will not be assessed.  
If a participant does not undergo surgery due to an apparent clinical complete response to neoadjuvant 
therapy, the participant will continue taking IP for 41 days after the decision to not operate. Quality of 
recovery and correlative outcomes will not be assessed; however, the participant will continue to follow 
all other study related procedures and visits with timing defined based on the decision to not operate.  
If a participant undergoes surgery without a liver resection or ablation due to an apparent clinical 
complete response, the participant will continue IP and all other study procedures as scheduled. 
If a participant undergoes surgery without a complete resection or ablation of all known disease due to 
progression, unresectable disease, or any reason other than a clinical complete response, the participant 
should discontinue IP as soon as possible. PFS, OS, the side-effect profile, and quality of recovery will be 
measured as scheduled. ctDNA and correlative outcomes will be measured once postoperatively.  
If a participant undergoes surgery with the resection of extrahepatic metastases discovered 
intraoperatively, and no known disease is left unresected or unablated, the participant should continue 
IP and all other study procedures as scheduled. 
If a participant is found to have additional hepatic or extrahepatic metastases during follow-up that in 
retrospect were present prior to surgery and were not removed at the time of surgery, the participant will 
discontinue IP as soon as possible. These participants will be monitored for AEs until 30 days after the last 
dose was administered. All other study procedures will continue as scheduled.  
If the final pathology from the liver resection is not consistent with metastatic colorectal carcinoma, the 
participant will discontinue IP as soon as possible and be monitored for AEs for 30 days after the last dose. 
PFS, OS, ctDNA, and correlative outcomes will not continue to be assessed. Quality of recovery will be 
evaluated as scheduled. . Note, if the medical care team and investigators believe the pathology result 
represents a pathological complete response to neoadjuvant therapy participants will continue to take IP 
until 41 days postoperatively and follow all study procedures.  
6.4.2 
Withdrawal 
Participants can choose to withdraw from the study at any time for any reason. As discussed above, 
participants who choose to discontinue IP or placebo will continue all study related procedures and visits 
if they are agreeable. For participants who withdraw and no longer wish to take part in any study related 
activities (including medical record review for long term outcomes), they will stop the IP as soon as 

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possible. If participants received any doses of IP or placebo prior to being withdrawn, they will be followed 
by study personnel for 30 days after the last dose to monitor for AEs. Withdrawn participants will be 
contacted by the research team to arrange an early termination visit, as detailed in Section 8.6. Every 
effort will be made to obtain permission to document the reason for withdrawal. Any data collected prior 
to the withdrawal may be retained and used by the sponsor. Participants who withdraw from the study 
prior to receiving study treatment  will not receive any further study specific tests and will not be included 
in any analysis.  These withdrawn participants will be replaced with another participant.  
 

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7 INTERVENTIONS  
7.1 
Investigational Product  
QBECO is a suspension of inactivated E. coli cells in physiological saline (0.9%). The concentration of cells 
in the suspension is normalized to an optical density (OD) of 5.0 ± 1.0 per mL when measured at 600 nm. 
The product has been developed by Qu Biologics as part of a novel class of microbial based 
immunotherapies, termed SSIs. QBECO is designed to activate a site specific innate immune response in 
the GI tract and related organs, including the liver.  
7.1.1 
Acquisition, Formulation and Packaging 
7.1.1.1 
Acquisition and Formulation 
The IP and placebo will be provided by the manufacturer, Qu Biologics. These will be formulated by the 
manufacturer in an injectable sodium chloride suspension and will require no additional preparation. 
7.1.1.2 
Packaging 
The IP and placebo will be packaged in 3 mL, EZ-Fill Borosilicate vials that conform to USP Type I 
requirements with 13-mm “Flurotec” (fluorinated polymer)-coated, bromobutyl rubber stoppers and 
TrueEdge flip off seals. Each vial will contain 2.3 mL of IP or placebo.  
QBECO SSI is a parenteral preparation of inactivated whole-cell bacteria in normal saline. The suspension 
should be agitated gently before each use to ensure uniform distribution. A multi-dose vial adapter is 
supplied with each study drug vial, allowing for repeated puncture and withdrawal. For first use of each 
vial, the multi-dose vial adapter is inserted and then will remain in place. Prior to each dose, the rubber 
area of the multi-dose vial adapter is swabbed with an alcohol swab and then the Luer-Lok syringe is 
attached, and the dose withdrawn. The Luer-Lok syringe is then detached from the multi-dose vial adapter 
and the needle attached to the syringe. A maximum of 10 doses (i.e., 20 days of treatment) can be 
withdrawn from each vial. 
7.1.2 
Treatment Assignment Procedures 
Study intervention (QBECO vs. Placebo) will be assigned by the randomization process described in 
Section 6.3.1.  
7.1.3 
Dosage and Administration  
Participants will receive either QBECO or placebo, 0.1 mL delivered via subcutaneous injection (SC) once 
every 2 days (q2days), for 11 to 120 days preoperatively and 41 days postoperatively.  
The first preoperative study treatment dose must not be given within 14 days following completion of 
neoadjuvant chemotherapy (the last dose of chemotherapy must be at least 14 days prior to the first dose 
of IP). Chemotherapy can cause abnormalities in laboratory tests of hematologic and immune function, 
which would complicate the ability to determine differences in efficacy or toxicity that are related to the 
IP. Hematologic and immune function generally returns to baseline by 14 days following completion of 
chemotherapy. Participants are intended to receive a minimum of 6 doses of the study treatment 

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preoperatively (minimum of 11 days of treatment). Therefore, the last dose of neoadjuvant chemotherapy 
must be at least 25 days prior to surgery.  
The last preoperative dose is intended to be given 1 day before surgery. Given the q2day dosing regimen, 
some participants will be scheduled to receive their last dose 2 days prior to surgery. These participants 
will be asked to administer an additional dose 1 day before surgery (back-to-back doses). 
Participants will receive the maximum number of preoperative doses within the constraints of the criteria 
listed above. The timeline from consent for surgery to operation ranges from approximately 14-28 days, 
which would allow 7-14 doses of the IP preoperatively. Participants with delayed operations will continue 
the IP or placebo until surgery for a maximum of 120 days. In the unlikely event that surgery is delayed 
beyond the 120-day maximum, the participant will be withdrawn as specified in Section 6.4.1. 
Postoperative treatment with IP will start on POD1 and continue every 2 days until POD41.  
7.1.4 
Procedures for Intervention Training 
Study treatment will be self-administered by the participant. Participants will receive SC injection teaching 
by a study team member or health care professional before receiving the IP or placebo. Participants may 
delegate one or more individuals who can assist with injections.  
7.1.5 
Dose Modification  
A local skin immune response (LSIR) (i.e., pink/red erythema at the injection site) may occur within the 
first 24 hours after injection. The LSIR may be mildly tender to the touch but not painful and slightly raised 
but not swollen. The LSIR is comparable to the skin wheal associated with a positive tuberculosis skin test 
and is indicative of a local innate immune response.  
In the event of a local skin immune response involving erythema approximately 7.0 cm or greater and/or 
interfering with normal functions of daily life, QBECO SSI dose will be reduced to 0.05 mL. If a local skin 
immune response involving erythema approximately 7.0 cm or greater and/or interfering with normal 
functions of daily life occurs following the reduced 0.05 mL dose, QBECO SSI dose will be further 
reduced to 0.02 mL. If the LSIR still meets the above criteria after 2 additional doses at the 0.02 mL dose, 
treatment will be stopped. 
In the unlikely event of a skin reaction involving edema, induration and/or erythema approximately 
10 cm or greater, treatment will be discontinued until the skin reaction resolves, at which time 
treatment can be continued with a 0.02 mL dose. 
Between study visits, participants will be advised to contact the research team if they experience a LSIR 
meeting the above criteria. The dose may be held until an in person assessment is able to be performed 
at the investigators discretion if required. Any dose modifications must be approved by a study 
investigator. 
Subjects receiving QBECO SSI may experience mild, transient fatigue that will resolve within a few days of 
initiating treatment. Study treatment does not need to be modified or discontinued in this context.   

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7.1.6 
Prior and Concomitant Medications/Treatments 
Drug interactions are not expected to occur with study IP. For participants receiving neoadjuvant 
chemotherapy, the last dose of chemotherapy must be at least 14 days prior to the first dose of IP and at 
least 25 days prior to surgery to ensure that hematologic and immune function are not compromised by 
chemotherapy while participants receive IP. 
Adjuvant chemotherapy, if planned, should likewise not be administered until completion of the IP. 
7.1.7 
Assessment of Participant Compliance  
Compliance will be assessed according to the Schedule of Procedures in Section 8. Should a participant be 
found non-compliant with administering the IP or placebo, every reasonable effort will be made to 
improve compliance, such as offering additional injection teaching. Non-compliant participants will still 
be included as per the principles of intention-to-treat analysis and will be encouraged to continue the 
usual follow-up schedule. 
7.1.8 
Receiving, Storage, Dispensing and Return 
7.1.8.1 
Receipt of Investigational Product 
The IP manufacturer, Qu Biologics, will ship IP and placebo to participating centres. Products will be 
maintained at appropriate storage temperatures of 2-8° C throughout transport. 
Upon receipt of the IP or placebo, an inventory must be performed and a receipt log filled out and signed 
by the research team member accepting the shipment. Any damaged or unusable product in a given 
shipment will be documented in the study files. The site must notify the Coordinating Centre of any 
damaged or unusable product that was supplied to the site. The Coordinating Centre will subsequently 
notify the sponsor. 
7.1.8.2 
Storage and Stability 
Study IP will be stored at 2-8° C in a secure facility at each participating site, accessible to authorized study 
personnel. Participants will be instructed to store vials in the refrigerator to maintain the appropriate 
storage temperature once dispensed. 
7.1.8.3 
Dispensing of Investigational Product 
IP or placebo and injection supplies will be dispensed to participants by authorized study personnel. The 
dispensing study personnel will provide instructions for safe storage and use.  
While admitted to hospital, site leads will be responsible for ensuring the study drug is available to the 
nursing team, stored correctly, and that administration orders are entered into the participant’s medical 
chart if needed. Study drug may be administered by the nursing staff or the participant, based on local 
site arrangements and requirements. Upon discharge, study personnel will be responsible for revisiting 
administration instructions, as needed. 
The IP and placebo will be reconciled by the research team at regular intervals, in addition to 
reconciliations performed during monitoring. Reconciliation will include verification of IP or placebo kit 

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assignment, inventory, and dispensing documentation. Participants will keep diaries to document the 
dates of IP administration. 
7.1.8.4 
Return and/or Destruction of Investigational Product 
Participants will be informed about the need to return all unused IP or placebo to the dispensing 
personnel. At the completion of the study, there will be a final reconciliation of IP shipped, used, and 
remaining. This reconciliation will be documented. Any discrepancies will be investigated, resolved, and 
documented prior to return or destruction of unused product. Documentation of product destroyed on 
site will be retained in the study files. 
 
 
 

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IP: investigational product, POD: post-operative day, ctDNA: circulating tumour DNA, CEA: carcinoembryonic antigen, CBC: complete blood count, OS: overall survival, PFS: progression free survival, QoR: 
quality of recovery 
*  Blood samples will be drawn only for participants who are still in hospital. A protocol deviation will not be required for blood samples that were unable to be obtained on the day of discharge 
a. Compliance assessment and side effect profile can also be collected the day before surgery via telephone if unable to collect the information on the day of surgery   
b. When appropriate (see Section 8.4) 
c. For specific imaging requirements, see Section 8.3 
d. Adverse events will be monitored at each contact with a participant until at least 30-days following the last treatment dose. This time may be rounded to the next visit. Extended follow-up applies in the 
event of pregnancy (Section 9.6) 
e. Following recurrence, no additional imaging or laboratory evaluations will be performed, however participants will continue to be followed for OS 
f. Data can also be collected upon review of medical records if available. If data is not available in participants medical records, study centre to perform outlined tests in table above 
g. ± 30 days of site’s awareness 
 
 
8 STUDY SCHEDULE AND PROCEDURES 
Participants will be followed from screening until 5 years after enrollment. Data may be collected in person, via phone, email, or online survey as applicable. 
Please see below for the schedule of study assessments: 
 
Table 1. Schedule of Procedures: Up to Day of Surgery 
 
  
Prior to surgery 
 
 
Day of surgery  
Prior to randomization 
Before starting IP, after chemotherapy (if 
applicable) 
Q2days,  
11-120 days pre-op 
Eligibility Assessment and Informed Consent 
x 
  
  
  
Demographic Information 
x 
 
 
 
Routine Bloodwork 
  
x 
  
  
Pregnancy Testb 
X 
  
  
  
Intervention 
  
  
x 
  
Compliance Assessment 
  
  
  
Xa 
Imagingc 
x 
  
  
  
CEA and CBC 
  
x 
  
  
PFS and OS Assessment 
  
  
  
  
ctDNA 
  
x 
  
x 
Side-effect profiled 
  
  
  
Xa 
QoR Survey 
  
x 
  
  
Correlative Endpoints 
  
x 
  
x 
 
 
 
 
 
 
 
 

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IP: investigational product, POD: post-operative day, ctDNA: circulating tumour DNA, CEA: carcinoembryonic antigen, CBC: complete blood count, OS: overall survival, PFS: progression free survival, QoR: 
quality of recovery 
*  Blood samples will be drawn only for participants who are still in hospital. A protocol deviation will not be required for blood samples that were unable to be obtained on the day of discharge 
a. Compliance assessment and side effect profile can also be collected the day before surgery via telephone if unable to collect the information on the day of surgery   
b. When appropriate (see Section 8.4) 
c. For specific imaging requirements, see Section 8.3 
d. Adverse events will be monitored at each contact with a participant until at least 30-days following the last treatment dose. This time may be rounded to the next visit. Extended follow-up applies in the 
event of pregnancy (Section 9.6) 
e. Following recurrence, no additional imaging or laboratory evaluations will be performed, however participants will continue to be followed for OS 
f. Data can be collected upon review of medical records if available. If data is not available in participants medical records, study centre to perform outlined tests in table above 
g. ± 30 days of site’s awareness 
 
 
 
 
 Table 2. Schedule of Procedures: After Surgery 
 
 
 
 
 
 
  
 
 
 
 
 
 
Q2 
days, 
POD1
-41 
POD1
* 
POD4* 
(± 1 
day) 
 
6 weeks 
post-op 
(± 10 
days) 
3 
months 
post-op 
(± 14 
days) 
6 
months 
post-op 
(± 28 
days) 
9 
months 
post-op 
(± 28 
days) 
12 
months 
post-op 
(± 28 
days) 
15 
months 
post-op 
(± 28 
days) 
18 
months 
post-op 
(± 28 
days) 
21 
months 
post-op 
(± 28 
days) 
24 
months 
post-op 
(± 28 
days) 
30 
months 
post-op 
(± 28 
days)  f 
36 
months 
post-op 
(± 28 
days)  f 
42 
months 
post-op 
(± 28 
days)  f 
48 
months 
post-op 
(± 28 
days)  f 
54 
months 
post-op 
(± 28 
days)  f 
5 years 
post-op 
(± 28 
days) 
‘Disease 
Progression 
e(±30 days) g 
Eligibility 
Assessment 
and 
Informed 
Consent 
  
 
 
 
  
  
  
  
  
  
  
  
 
 
 
 
 
  
  
Demographic 
Information 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Routine 
Bloodwork 
  
 x 
x 
 x 
  
  
  
  
  
  
  
  
 
 
 
 
 
  
  
Pregnancy 
Testb 
  
  
  
  
  
  
  
  
  
  
  
  
 
 
 
 
 
  
  
Intervention 
x 
x 
x 
x 
  
  
  
  
  
  
  
  
 
 
 
 
 
  
  
Compliance 
Assessment 
  
  
  
  
x 
  
  
  
  
  
  
  
 
 
 
 
 
  
  
Imagingc 
  
  
  
  
x 
x 
x 
x 
x 
x 
x 
x 
X  
X  
X  
X  
X  
x 
  
CEA and CBC 
  
  
  
  
x 
x 
x 
x 
x 
x 
x 
x 
X 
X 
X 
X 
X 
x 
  
PFS and OS 
Assessmente 
  
  
  
  
x 
x 
x 
x 
x 
x 
x 
x 
X  
X  
X  
X  
X  
x 
  
ctDNA 
  
x 
x 
x 
  
x 
  
x 
  
x 
  
x 
 
 
 
 
 
  
x 
Side-effect 
profiled 
  
  
  
x 
x 
  
  
  
  
  
  
  
 
 
 
 
 
  
  
QoR Survey 
  
  
x 
x 
  
  
  
  
  
  
  
  
 
 
 
 
 
  
  
Correlative 
Endpoints 
  
x 
x 
x 
  
x 
  
x 
  
x 
  
x 
 
 
 
 
 
  
x 

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Protocol Number: 4023 
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8.1 
Prior to Surgery 
Participating sites will pre-screen patients for eligibility according to the inclusion and exclusion criteria in 
Section 6.1 and 6.2 and site approvals. Should a patient be identified as a potential candidate for 
enrollment, a member of the circle of care will obtain permission from the patient to introduce a research 
team member. The research team will obtain informed consent and then confirm eligibility. If eligibility 
cannot be confirmed due to missing imaging, these will be obtained by the surgeon prior to enrollment. 
Once eligibility is confirmed, the research team will randomize the participant. 
Demographic information including but not limited to age, sex and ethnicity will be collected once consent 
is received. Baseline blood-based outcome measures and routine preoperative bloodwork will be drawn 
prior to starting the IP or placebo. The blood-based outcome measures are described in Section 4.3 and 
4.4. Routine preoperative bloodwork will include the following: complete blood count (CBC), serum 
chemistry, serum creatinine, liver enzymes and liver function tests (LFTs), coagulation profile (INR), serum 
carcinoembryonic antigen (CEA), and complete blood count (CBC). All bloodwork will be drawn within 28 
days prior to starting the IP.   
8.2 
Day of Surgery 
Research staff will record any missed preoperative doses of the study drug, as well as any AEs that a 
participant may have experienced. This information can also be collected the day before surgery via 
telephone.  
Select blood-based outcome measures will be re-evaluated prior to skin incision on the day of surgery. A 
sample of fresh tumour tissue will be obtained from a representative hepatic tumour and from the 
primary colorectal tumour, if resection occurs at the same operation as the liver resection, along with a 
sample of normal adjacent tissue (one of each type per surgical specimen); to be used for creating the 
ctDNA assay and molecular profiling of the tumour microenvironment. The ideal size for each sample 
will be > 1cm3. The surgical specimens will be handled fresh and fixed in 10% buffered formalin and then 
transferred to 70% ethanol. Subsequently, the fixed specimen will be shipped to Ottawa for processing 
and for tumor cellularity assessment. If no suitable tumour tissue is found (i.e.,  < 30% tumour cellularity 
or insufficient DNA extraction), archival formalin-fixed paraffin-embedded (FFPE) tissue from the 
participant will be obtained through pathology. If FFPE tissue is not available, tissue slides (20 unstained 
and 2 H&E stained slides) will be obtained instead. Tissue from a prior resected or biopsied specimen 
from the participant may also be sought if archival tissue from the resected specimen is still inadequate.  
A study site may opt out of fresh specimen procurement. In this case, archival slides from the surgical 
resection of the liver, that meet acceptable tumor cellularity (> 30% cellularity) will be requested.  
Some participants enrolled at the Ottawa Hospital may also undergo one additional blood draw for 
measurement of ctDNA postoperatively, on the day of surgery. 
8.3 
After Surgery 
The participant will continue to receive the IP or placebo for 41 days postoperatively, starting on POD1.  

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24 
 
Postoperative bloodwork values will be recorded on POD1, POD4±1 if they are still in hospital including: 
CBC, serum chemistry, serum creatinine, liver enzymes and LFTs, and coagulation profile. The participant 
will only be required to undergo research bloodwork on POD1 and 4±1 if they are still in hospital at these 
time periods. At 6-weeks post-operatively, blood-based outcome measures and routine bloodwork will 
be drawn. The blood-based outcome measures are described in Section 4.3 and 4.4. Routine bloodwork 
will include the following: CBC, serum chemistry, serum creatinine, liver enzymes and LFTs, and 
coagulation profile (INR). 
Visits may be virtual (i.e., by phone, email, online survey, review of medical records as applicable) after 2 
years. 
If the participant is identified as having disease progression, select blood-based outcome measures will 
also be redrawn at that time of sites awareness. Participants will stop taking the IP and no further study-
protocolled imaging or laboratory investigations will be required after disease progression, however the 
participant will continue to be followed for OS.  
For assessment of disease progression, serial CT scans of the chest, abdomen, and pelvis (or CT chest and 
MRI abdomen), and serum CEA and CBC should be performed every 3 months for 2 years postoperatively, 
then every 6 months for 5 years postoperatively. After 2 years, clinicians may follow their routine imaging 
and biochemical surveillance regimens. Imaging will ideally be performed at the treating study centre, 
however if this is not possible, outside images will be accepted. 
8.4 
Pregnancy Testing 
Women of childbearing potential (WOCBP) must undergo a pregnancy test prior to randomization and 
within 30 days prior to starting the IP or placebo. Both male and female patients will be informed of 
contraceptive requirements and must agree to these requirements as part of confirming eligibility. The 
definition of WOCBP and the specific contraceptive requirements are detailed in Appendix B.  
8.5 
Pregnancy Visit and Follow-Up  
WOCBP and fertile male participants will be required to use a highly effective contraceptive while 
receiving IP or placebo, as specified in Appendix B. Participants who become pregnant after commencing 
the IP or placebo will be asked to discontinue the product however will remain enrolled.  
The following groups will be followed until 6 to 8 weeks beyond the date of delivery or the termination of 
the pregnancy: 
 
Participants who become pregnant while receiving IP  
 
Partners of male participants who become pregnant while the male participant receives IP. 
Follow-up will be contingent upon investigators obtaining informed consent from the 
pregnant female partner to record pregnancy information. 
AEs during this extended follow-up period will be defined and recorded according to Section 9.6. 
The study sponsor will be informed of pregnant participants and pregnant partners of male participants 
within 24 hours of when investigators are first notified. 

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25 
 
8.6 
Early Termination Visit 
Participants who withdraw from the study prior to study completion will be asked to complete an early 
termination visit with a study team member, with the exception of participants who withdrew prior to 
receiving IP (these participants will not complete any further study procedures and will be replaced). At 
this visit, the following data will be recorded: compliance with IP injections (if applicable), reason for 
discontinuation of IP (if applicable), reason for withdrawal, disease progression status, and AEs. 
Participants who discontinue the study drug will also be asked if they would agree to ongoing follow-up 
as per the study protocol.  
8.7 
Final Study Visit 
The final study visit will be at 5 years postoperatively, at which time disease progression and OS will be 
documented. At the conclusion of the trial, survival data may be abstracted from the participant’s medical 
record beyond 5 years for the sake of time-to-event analysis, however this would not necessitate a formal 
appointment.  
8.8 
Protocol Deviations 
It is the responsibility of the investigators to ensure that only investigative procedures, as outlined in this 
protocol, are performed on study participants; the occurrence of deviations from the protocol or standard 
operating procedures (SOPs) are limited; and compliance with the regulations is maintained. Planned 
deviations from the protocol must not be implemented without prior agreement from the sponsor and 
approval from the local research ethics boards (REBs), as required, unless to eliminate an immediate 
hazard to a participant.  
Planned or unplanned deviations may occur on the part of the participant, the investigators, or the study 
research team. Following a deviation, corrective/preventative actions are to be developed and 
implemented in a timely manner. Protocol deviations will be documented and reported as required and 
assessed where necessary during analysis. 
8.9 
Handling Procedures for Blood and Tissue Derived Outcome Measures 
Biologic materials will be stored appropriately for transport and disposed of according to institutional 
standards following analysis. A correlative sample manual of Standard Operative Procedures will be 
provided to each site. All correlative samples will be sent to the lab of Dr. Rebecca Auer at the Ottawa 
Hospital Research Institute. Foundation Medicine (www.foundationmedicine.com) will provide materials 
for the measurement of ctDNA, and NKMAX (www.nkmax.com/eng/) will provide testing kits for one of 
the NK cell functional assays, if these are being performed. 
 
 

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9 ASSESSMENT OF SAFETY 
The safety of research participants is foremost and should always be considered throughout the conduct 
of research.  
9.1 
Definitions 
9.1.1 
Adverse Events 
An AE is any untoward medical occurrence in a participant who has been administered a pharmaceutical 
product and does not necessarily need to have a causal relationship with the product.  
To be considered an AE, the medical occurrence must occur within the AE recording period. The recording 
period is after the first administered dose of IP and  30-days after the last administered dose. Events 
starting outside this period will not be considered AEs. 
Postoperative complications will be recorded as AEs if they occur within the AE recording period. 
Postoperative complications have been defined in Section 4.3. Depending on severity, a postoperative 
complication may qualify as a SAE. 
The following events will not be considered AEs and therefore do not require recording:  
 
Manifestations of pre-existing diseases unless, at the discretion of the investigators, the disease 
or condition worsens in severity or frequency during the recording period. 
 
Common events occurring within the postoperative period. The postoperative period will be 
measured from the time of surgery to either the time of discharge or POD28, whichever occurs 
later. However, a common event within the postoperative period may be recorded as an AE if (1) 
at the discretion of the investigators, the event has a severity or duration that is not in keeping 
with the normal postoperative course AND (2) the event occurs within the AE recording period. 
Common events within the postoperative period include but are not limited to: 
o Abdominal pain 
o Nausea 
o Vomiting 
o Obstipation 
o Fatigue 
o Headache 
o Pruritus 
o Weight loss (less than 15% of the pre-surgical weight) 
o Bleeding per rectum if a bowel resection was performed at the time of surgery 
o Urinary retention 
o Difficulty ambulating 
 
Elective medical or surgical procedures 
9.1.2 
Serious Adverse Events 
A SAE or reaction is any untoward occurrence that meets at least one of the following criteria: 

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 
An event that results in death. 
 
An event that is life-threatening. This is defined as events where the participant is at risk of death; 
it does not refer to an event which hypothetically might have caused death if it were more severe. 
 
An event that requires inpatient hospitalization or prolongation of existing hospitalization. 
Prolonged hospitalization will be defined as lasting  21 days from the date of surgery. 
 
Results in persistent or significant disability/incapacity. 
 
A congenital abnormality in a child of a participant or partner of a male participant who delivered 
after the start of IP. 
 
A spontaneous abortion in a participant or partner of a male participant who delivered after the 
start of IP. 
9.1.3 
Unexpected Adverse Event 
An unexpected AE is any AE that is not identified in nature, severity, or frequency in the current IB. 
9.1.4 
Adverse Drug Reaction and Unexpected Adverse Drug Reaction 
An ADR is any noxious or unintended response to a medicinal product related to any dose.  
The phrase "responses to a medicinal product" means that a causal relationship between a medicinal 
product and an AE is at least a reasonable possibility, i.e., the relationship cannot be ruled out.  
The expression “causal relationship” is meant to convey that in general there are facts, evidence, or 
arguments to suggest a reasonable causal relationship.  
All serious and unexpected ADRs will have expedited reporting to regulatory agencies in accordance with 
the ICH-GCP and local regulatory requirements. 
9.2 
Assessment of an Adverse Event 
9.2.1 
Relationship (Causality/Relatedness) 
The causality assessment is the determination, according to the investigator’s clinical judgment, of the 
existence of a reasonable possibility that the IP caused or contributed to an AE.  
If the investigator or delegated sub-investigator is unsure about whether the study drug caused or is 
related to the AE, then the event will be handled as “related” for reporting purposes. If the causality 
assessment is "unknown but not related” to the study drug, this should be clearly documented in the 
source documents.  
9.2.2 
Expectedness  
Events are classified as unforeseen or unexpected if the nature, severity, or frequency is not consistent 
with the risk information set out in the IB.     
9.2.3 
Seriousness 
Events are classified as serious if they are associated with effects threatening the life or physiological 
functions of a participant. Refer to the definition for “Serious Adverse Events” in Section 9.1.2. 

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9.2.4 
Severity  
The term "severe" is often used to describe the intensity (severity) of a specific event (e.g., mild, 
moderate, or severe myocardial infarction). However, the event itself may be of relatively minor medical 
significance (such as severe headache). This is not the same as "serious," which is based on 
participant/event outcome or action criteria usually associated with events that pose a threat to a 
participant's life or functioning. The terms “serious” and “severe” are not synonymous. Seriousness (not 
severity) serves as a guide for defining regulatory reporting obligations. 
To assess the severity of an AE the investigators will use the NCI Common Terminology Criteria for Adverse 
Events (CTCAE v5.0). 
9.3 
Adverse Event Recording 
Investigations into potential AEs should be done during each contact with a participant within the AE 
recording period. The recording period is after the first administered dose of IP and  30-days after the 
last administered dose. The last AE assessment may be rounded to the next visit if there is not a scheduled 
encounter 30-days after the last dose of IP.  
Investigations may be done through specific questioning and by examination as appropriate. Information 
on all AEs should be recorded promptly in the source document and assessed by an investigator in a timely 
manner allowing sufficient time to meet required reporting timelines for SAEs and serious and unexpected 
adverse drug reactions (SUADRs) if needed. AE case report forms (CRFs) should be completed using source 
documents by a delegated research team member in a timely manner. All clearly related signs, symptoms, 
and abnormal diagnostic procedures should be recorded in the source document and should be grouped 
under one diagnosis. Each diagnosed AE should then be categorized in accordance with Medical Dictionary 
for Regulatory Activities (MedDRA) classifications/the revised NCI CTCAE v5.0.  
9.4 
Reporting of SAEs and Unanticipated Events 
9.4.1 
Investigator reporting: Notifying the Research Ethics Board 
SAEs and unanticipated events should be recorded and reported to the REB in accordance with local 
reporting requirements and timelines. 
9.4.2 
Investigator reporting: Notifying the Coordinating Centre 
The investigators are responsible for reporting SAEs and SUADRs to the Coordinating Centre within the 
following timelines: 
 
All deaths and immediately life-threatening events, whether related or unrelated, will be 
recorded and reported to the Coordinating Centre within 24 hours of site awareness.  
 
SAEs other than death and immediately life-threatening events, regardless of relationship, 
will be reported to the Coordinating Centre within 72 hours of site awareness.  
AE information will be entered into the CRF in a timely manner and no later than 15 days from the time 
the investigator becomes aware of the event. 

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SAE information will be entered into the CRF in a timely manner from the time the investigator becomes 
aware of the event. 
Events that are assessed to be serious, unexpected, and related, or cannot be ruled out as related, to the 
IP are considered SUADRs. Reporting for SUADRs should include a description of the event in sufficient 
detail to allow for a complete medical assessment of the case and independent determination of possible 
causality. The minimum information required includes at least one identifiable participant, one 
identifiable reporter, one serious reaction, and one suspect product.  
Additionally, a Suspect Adverse Reaction Report (Council for International Organizations of Medical 
Sciences (CIOMS) I Form, see Appendix A) must be completed by the investigator and forwarded to the 
Coordinating Centre within 24 hours of site awareness. Information on other possible causes of the event, 
such as concomitant medications and illnesses should also be provided as soon as is made available. 
9.4.3 
Coordinating Centre Reporting of SUADRs: Notifying Health Canada 
The regulatory sponsor is responsible for reporting SUADRs to regulatory authorities in accordance with 
local expedited reporting requirements and timelines. These activities have been delegated to the 
Coordinating Centre. In addition, the Coordinating Centre will complete the ADR Expedited Reporting 
Summary Form and submit this form in conjunction with the completed CIOMS Form to the appropriate 
Health Canada directorate. 
9.4.4 
Coordinating Centre Reporting of SUADRs: Notifying Sites 
The Coordinating Centre is responsible for distributing blinded expedited reports of SUADRs to each 
investigator for submission to local REBs within 15 days of Coordinating Centre awareness. 
9.4.5 
Coordinating Centre Reporting of SAEs and SUADRs: Notifying the Sponsor 
The Coordinating Centre will report all SAEs and SUADRS to the regulatory sponsor (Qu Biologics) within 
5 days of Coordinating Centre awareness for fatal and life-threatening events and 10 days of awareness 
for other non-fatal events. SUADRS will be sent to the regulatory sponsor prior to submission to Health 
Canada. 
9.5 
Duration of Follow-up for Adverse Events  
AEs occurring after the first administered dose of the IP and 30-days after the last administered dose will 
be collected. AEs recorded during this period will be followed through to resolution, or until the event is 
assessed as chronic or stable. 
9.6 
Pregnancy and Adverse Events 
Extended monitoring for AEs will be required for the following groups: 
 
Participants who become pregnant while receiving IP  
 
Partners of male participants who become pregnant while the male participant receives IP 
These participants and partners will be followed until 6 to 8 weeks after delivery or 
termination of the pregnancy. For partners, follow-up is contingent upon investigators 

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receiving informed consent collect relevant health and pregnancy information. The AE 
recording period for these participants and partners will therefore be extended to the last 
pregnancy-related follow-up encounter.  
Any pregnancy complication, elective termination, or congenital malformation will be considered AEs. AEs 
may also be considered SAEs if they meet the definition specified in Section 9.1.2. Spontaneous abortions 
and congenital malformations will be considered SAEs and will be reported as such. 
9.7 
Data and Safety Monitoring Board 
A Data and Safety Monitoring Board (DSMB) will be established for this trial. The role of the DSMB will be 
to review safety data over the study duration. The specific responsibilities, procedures, and meeting 
format of the DSMB is detailed in the DSMB Charter.  
 

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10 SITE MONITORING, auditing, and inspecting 
10.1 Site Monitoring Plan 
Site monitoring is conducted to ensure the safety of human study participants and the protection of their 
rights and well-being. Monitoring also verifies that collected study data is accurate, complete, and 
verifiable by source documentation and that the study is conducted in accordance with the protocol and 
operating procedures. 
Monitoring for this study is the responsibility of the sponsor. The delegated monitor will evaluate study 
processes and documentation based on the approved protocol/amendment(s), Part C, Division 5 of the 
Food and Drug Regulations, the ICH-GCP, and institutional policies. 
The extent and nature of monitoring is outlined in the Monitoring Plan. The monitoring plan specifies the 
frequency of monitoring, monitoring procedures, level of site monitoring activities (e.g., the percentage 
of participant data to be reviewed), and distribution of monitoring reports. Monitoring activities may be 
performed in person and/or remotely. Reports of findings identified during monitoring activities will be 
provided to sites detailing any required actions. Documentation of monitoring activities and findings will 
be provided to the site study team and the study principal investigators (PIs). The institution and/or local 
REB reserves the right to conduct independent audits as necessary. 
The investigators are responsible for ensuring monitors and/or quality assurance reviewers are given 
access to all study-related documents noted above and study related facilities (e.g., pharmacy, diagnostic 
laboratory, etc.), and have adequate space to conduct the monitoring visit or audit. 
10.2  Auditing and Inspecting 
The investigators will provide direct access to source data/documents for the purposes of study-related 
monitoring, audits, and inspections by the coordinating centre, REBs, the sponsor, and applicable 
regulatory bodies. The investigator will permit the review of all study related documents (e.g., source 
documents, regulatory documents, data collection instruments, study data, etc.) and will ensure access 
to applicable study-related facilities (e.g., pharmacy, diagnostic laboratory, etc.). 
 

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11 STATISTICAL CONSIDERATIONS 
11.1 Study Hypotheses 
We hypothesize that participants receiving QBECO will experience a 65% improvement in the percentage 
with PFS compared to those receiving placebo.  
Sample Size Considerations 
Given a predicted 40% at 2-years in participants undergoing potentially curative resection of colorectal 
liver metastases,22 a 65% improvement in the percentage surviving progression free represents a PFS of 
approximately 1.65*40% = 65% at 2 years.  Under the assumption of exponential survival, these two-year 
PFS percentages imply an annual rate of events in the control group of 0.458 and a rate in the QBECO 
group is 0.215; the corresponding hazard ratio (HR) for treatment is 0.47.  The table below shows a power  
to detect this hazard ratio of 0.47 (in a one-sided test, of the null hypothesis that HR=1 with alpha=0.05), 
and hazard ratios of 0.5 to 0.6 when the analysis is done once a pre-set number of study events 
(progression or death) has been observed.  We choose 54 events as our target, which gives 87% power 
for a 65% increase in survival at 2 years (HR=0.47) and 82% power for HR=0.5. 
 
Power (%) 
Number of Events 
HR = 0.47 
HR = 0.5 
HR = 0.55 
HR = 0.6 
51 
85 
80 
69 
57 
52 
86 
80 
70 
58 
53 
86 
81 
70 
58 
54 
87 
82 
71 
59 
55 
88 
82 
72 
60 
56 
88 
83 
72 
61 
It is anticipated that 58 participants can be recruited per year. Under the assumed event rates above, 
assuming an enrollment of 116 patients over two years, it is expected that the trial will reach 54 events  
at approximately 3 years after the first patient is enrolled.  These calculations are based on an expected 
10% of patients not undergoing liver resection due to progression of disease and 5% being lost to follow-
up or withdrawing for other reasons after surgery but before reaching the study endpoint (so that there 
are sufficient events in participants who undergo liver resection for an analysis in just that subset – the 
sensitivity analysis in section  11.3 ).  Projected enrolment will be approximately 20 participants for each 
of the participating sites.  
 
Planned Safety Analyses  
Two planned safety analyses of all safety data will be performed once 20 participants have had 6 weeks 
of follow-up and once 50 participants have had 6 weeks of follow-up. The DSMB will review the results 
of these safety analyses. Additional safety analyses may be reviewed by the DSMB as needed. 
11.2 Stopping Rules 
This study will be stopped prior to its completion if: (1) the intervention is associated with AEs that call 
into question the safety of the intervention; (2) difficulty in study recruitment or retention will significantly 

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impact the ability to evaluate the study endpoints; (3) any new information becomes available during the 
trial that necessitates stopping the trial; or (4) other situations occur that might warrant stopping the trial. 
11.3 Final Analysis Plan 
The cohorts for analysis will be defined as follows and are summarized according to clinical scenario in 
Appendix C:: 
1. Primary analysis cohort: This will be an intention-to-treat (ITT) cohort for the analysis of the 
primary and secondary outcomes. This cohort will analyze all enrolled participants who received 
at least one dose of IP according to treatment allocation. Withdrawn participants who received 
at least one dose of IP will be also included in this cohort, unless they withdraw consent to the 
use of their data.   
2. Correlative analysis cohort: This will be the cohort for the analysis of the correlative outcomes. 
This cohort will analyze all enrolled participants who undergo surgery, even if the surgery does 
not involve a liver resection or not all disease is resected or ablated 
3. Sensitivity analysis cohort: This will be a per-protocol cohort and only include participants who meet 
all of the following criteria. Note that withdrawn participants will be included if they meet the below 
criteria and do not withdraw consent for use of their data:  
- 
Receive a minimum of 80% of the prescribed preoperative doses of IP and at least 90% of the 
postoperative doses.  
- 
Undergo liver resection with or without ablation of all visible disease and pathology confirms 
CRLM or a pathologic complete response; or, undergoes surgery without a resection or 
ablation due to an apparent clinical complete response; or, does not undergo surgery due to 
an apparent clinical complete response. 
- 
No preoperative extrahepatic metastases are identified at any point. 
4. Safety analysis cohort: This cohort will include all enrolled participants who receive at least one 
dose of IP/placebo 
 
Descriptive statistics will be used to summarize variables in each treatment arm.  
OS and PFS of participants in each treatment arm will be evaluated using the Kaplan-Meier method and 
compared using the log-rank test. Participants will be censored at the time of last follow-up. Cox 
regression will be used to estimate hazard ratios and identify independent predictors of OS and PFS. The 
proportional hazards assumption will be assessed for all Cox regression models. Other outcomes will be 
evaluated using multivariable regression models appropriate for the type and distribution of the data.  
The amount and patterns of missing data and patients lost to follow-up will be evaluated. AEs, disease 
progression, or death may account for missing postoperative assessments. Participants with missing 
covariates will be excluded from multivariable models.  

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Additional hypothesis generating subgroup analyses will be performed according to the following disease 
and treatment characteristics: 
 
Synchronous vs. metachronous disease. Synchronous liver metastases will be defined as those 
identified before resection of the primary colorectal carcinoma, or those that in retrospect were 
present prior to resection of the primary, which is consistent with prior definitions.44,45 
Metachronous metastases will be defined as those identified following resection of the primary. 
 
Combination of surgery and ablation vs. surgery only. 
 
Progression identified at the time of surgery or prior to surgery. 
 
Some visible disease (primary or metastases) not resected or ablated at the time of surgery. 
 
Neoadjuvant vs. no neoadjuvant chemotherapy. 
 
Minor vs. major liver resection. Major resection will be defined as resection of 3 or more hepatic 
segments.46 

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12 DATA HANDLING AND RECORD KEEPING 
12.1 Confidentiality 
Information about study participants will be kept confidential and managed according to the 
requirements of the Personal Health Information Protection Act of 2004 (PHIPA) and the REB. PHIPA 
outlines the rules for the collection, use and disclosure of personal health information (PHI). The Act 
requires each participant to consent to the collection, use and access of PHI, unless consent is waived by 
the REB. Where consent is required, each participant must be informed of the following:  
 What PHI will be collected 
 Who will have access to their PHI information and why 
 Who will use or disclose their PHI 
 The rights of a participant to revoke their authorization for use of their PHI.  
If a participant revokes authorization to collect or use their PHI, the investigator may use all information 
collected prior to the revocation of participant authorization. For participants that have revoked 
authorization to collect or use PHI, attempts should be made to obtain permission to collect at least vital 
status (i.e., that the participant is alive) at the end of the scheduled study period. 
12.2 Source Documents 
Source data/documents are original documents, data, and records in a clinical study that are necessary 
for the reconstruction and evaluation of the study. Examples of these original documents and data records 
include, but are not limited to: 
 
Worksheets 
 
Hospital records  
 
Medical records 
 
Memorandum  
 
Participants’ diaries or evaluation checklists  
 
Pharmacy dispensing records  
 
Recorded data from automated instruments (i.e., ECGs) 
 
Copies or transcriptions certified after verification as being accurate and complete  
 
Participant files and records kept at the pharmacy  
 
Entries entered directly into the printed CRF 
Each participating site will maintain appropriate medical and research records for this study, in addition 
to regulatory and institutional requirements for the protection of participant confidentiality. If electronic 
source documents are printed, they should be signed and dated by the investigator to confirm content 
and subsequently filed with other source documents.  
The investigators and research team members listed on the Task Delegation Log (TDL) will have access to 
participant medical records and will collect only the information needed for the study. Sponsor delegated 
monitors, representatives of institutional committees, and regulatory authority representatives of the 
country in which the study is being conducted will also have access to examine records for the purposes 
of quality assurance reviews, audits, and evaluation of study safety. 

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12.3 Data Management Responsibilities 
Data collection and accurate documentation are the responsibility of the study personnel under the 
supervision of the investigators. All source documents and applicable laboratory reports should be 
reviewed as needed and used to ensure that data collected for the purposes of the study are accurate and 
complete. Contemporaneous review of laboratory results and the assessment of clinical significance for 
those results considered out of range should be documented by means of dated signature by the 
reviewing investigator. Study personnel, including data entry team members, should use source 
documents to complete CRFs.  
As part of the safety plan for this study, the investigator will review individual study participant records to 
ensure that appropriate mechanisms to protect the safety of study participants are being followed, that 
protocol requirements are being adhered to, and that data is accurate, complete, and secure. Participant 
records include, but are not limited to: consent forms, CRFs, data forms, laboratory specimen records, 
inclusion/exclusion forms, and medical charts. All study data will be collected by a member of the study 
research team and recorded in accordance with applicable procedures. 
12.4 Data Capture and Case Report Forms 
The study CRF is the primary data collection instrument for the study. Electronic/Paper CRFs will be used 
to collect data for this study. CRFs are to be completed by data capture personnel and signed off by an 
investigator in a timely manner. Good documentation practices should be implemented according to 
standard operating procedures. All data requested on the CRF must be recorded and verifiable by source 
document. 
12.5 Records Retention 
It is the responsibility of the REBs, investigators, and regulatory sponsor to retain study essential 
documents as per local regulatory requirements and GCP guidelines.   
Essential study documents must be maintained in a secure and confidential manner for participating 
Canadian sites for a period of 15 years. For the purposes of this study, the start date of the retention 
period is the date of the final report of the trial. Exceptions may be made for sites that close prematurely, 
wherein the start date for the retention period will be the date of notification to Health Canada of the 
sites closure. Sites conducting this study outside of Canada must maintain study records for the required 
retention period as stipulated by local regulatory authorities. All study records are then to be destroyed 
according to local and national policy and requirements. It is the investigators’ responsibilities to request 
authorization for destruction at the completion of the retention period and/or for the sponsor to inform 
the investigators/institution when these documents may be destroyed.  
12.6 Clinical Trial Registration 
In accordance with Health Canada’s Notice “Registration and Disclosure of Clinical Trial Information, 
November 30, 2007”, the sponsor will be responsible for registering the study on Clinicaltrials.gov, a 
publicly available registry that conforms to international standards for registries. 

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13 QUALITY CONTROL AND QUALITY ASSURANCE 
As per ICH-GCP and local regulations, the sponsor is responsible for ensuring the implementation and 
maintenance of systems that support quality assurance and quality control.  
The study must be conducted in compliance with the study protocol and all data collected must be 
accurate and verifiable by source documents. For the purpose of monitoring and auditing by the 
coordinating centre and/or Sponsor, and inspection by regulatory authorities, the site will provide direct 
access to all study related source data/documents. The sponsor will verify that the study is conducted and 
data has been collected, documented (recorded), and reported in compliance with the protocol, GCP, and 
applicable regulatory requirements.  
Data for the study will be centrally stored and managed by the Centre for Clinical Trial Support (CCTS). To 
ensure the quality of study data, quality assurance and control systems will be implemented using 
validated electronic quality control checks within the electronic data capture system. These verification 
measures will identify missing data, inconsistencies, and/or data anomalies. Both electronic and manual 
queries will be generated for resolution and review by sites.   
Access to secure and validated electronic systems used for the purposes of this study will be controlled 
by the sponsor. Access will only be granted to individual research team members upon review of training 
and qualification and authorization by delegation of the investigators.   
Quality assurance and control measures will be implemented to ensure training for specific trial–related 
tasks beyond the usual scope of practice. 
 

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14 ETHICS CONSIDERATIONS 
14.1 Ethical Standard 
The investigators will ensure this study is conducted in accordance with the principles set forth in The 
Belmont Report: Ethical Principles and Guidelines for the Protection of Human Participants of Research, 
and codified in the Tri-Council Policy Statement and/or the ICH E6.  
14.2 Research Ethics Board  
The protocol, informed consent form (ICF), recruitment materials, and all participant materials will be 
submitted to the REB for review and approval. Approval of both the protocol and the consent form must 
be obtained before any participant is enrolled. Any amendment to the protocol will require review and 
approval by the REB before the changes are implemented in the study, unless to eliminate an immediate 
hazard.   
14.3 Consent  
Informed consent is a process that is initiated prior to the individual agreeing to participate in the study 
and continues throughout study participation. A consent form describing in detail the study procedures 
and risks will be reviewed with and given to each participant. Consent forms will be REB-approved, and 
the participant is required to read and review the document or have the document read to him or her. 
The investigators or a designee will explain the research study to the participant and answer any questions 
that may arise. The participant will sign the informed consent document prior to any study-related 
assessments or procedures. Participants will be given the opportunity to discuss the study with their 
surrogates or think about it prior to agreeing to participate. They may withdraw consent at any time 
throughout the course of the study. A copy of the signed informed consent document will be given to 
participants for their records.   
Prior to involvement in any study-related activities, consent must be obtained in writing for each 
participant using the current REB approved informed consent from. It is the responsibility of the 
investigator to ensure that all advertisements and written information, including the ICF, disseminated to 
participants has been approved by the local REB prior to use. The ethics approved ICF and any other 
written information, must be provided to each participant, allowing ample time to ask and have answered 
any questions prior to making a decision regarding participation. Neither an investigator nor study staff 
should unduly influence or coerce a participant to participate in the study.   
The ICF will be signed and dated by the participant and individual obtaining consent. The consent process 
will be documented in the clinical or research record.   
The original ICF, in its entirety, will be maintained by the site, and a complete copy of the signed ICF 
provided to the participant. The rights and welfare of the participants will be protected by emphasizing 
to them that the quality of their clinical care will not be adversely affected if they decline to participate in 
this study. 
The provision of consent is an ongoing process and should be maintained throughout the duration of the 
study. Participants may withdraw consent at any time throughout the course of the study. 

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15 PUBLICATION/DATA SHARING POLICY 
Authorship on study publications will adhere to the Uniform Requirements for Manuscripts Submitted to 
Biomedical Journals of the International Committee of Medical Journal Editors. These requirements state 
“Authorship credit should be based on: 
1. Substantial contributions to conception and design, acquisition of data, or analysis and 
interpretation of data. 
2. Drafting the article or revising it critically for important intellectual content. 
3. Final approval of the version to be published. 
Authors should meet conditions 1, 2, and 3.” Where journal policies permit, all study site investigators 
who played a contributing role in the trial, including to its accrual, will be included in an Acknowledgement 
section. 
 

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16 REFERENCES 
1.  Tsuchiya Y, Sawada S, Yoshioka I, et al. Increased surgical stress promotes tumor metastasis. 
Surgery. 2003;133(5):547-555. doi:10.1067/msy.2003.141 
2.  Tai LH, Zhang J, Scott KJ, et al. Perioperative influenza vaccination reduces postoperative metastatic 
disease by reversing surgery-induced dysfunction in natural killer cells. Clin Cancer Res. 
2013;19(18):5104-5115. doi:10.1158/1078-0432.CCR-13-0246 
3.  Tai LH, Alkayyal AA, Leslie AL, et al. Phosphodiesterase-5 inhibition reduces postoperative 
metastatic disease by targeting surgery-induced myeloid derived suppressor cell-dependent 
inhibition of Natural Killer cell cytotoxicity. Oncoimmunology. 2018;7(6):e1431082. 
doi:10.1080/2162402X.2018.1431082 
4.  Tai LH, de Souza CT, Bélanger S, et al. Preventing postoperative metastatic disease by inhibiting 
surgery-induced dysfunction in natural killer cells. Cancer Res. 2013;73(1):97-107. 
doi:10.1158/0008-5472.CAN-12-1993 
5.  Seth R, Tai LH, Falls T, et al. Surgical stress promotes the development of cancer metastases by a 
coagulation-dependent mechanism involving natural killer cells in a murine model. Ann Surg. 
2013;258(1):158-168. doi:10.1097/SLA.0b013e31826fcbdb 
6.  Ananth AA, Tai LH, Lansdell C, et al. Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated 
Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence. PLoS One. 2016;11(5):e0155947. 
doi:10.1371/journal.pone.0155947 
7.  Gabrilovich DI. Myeloid-Derived Suppressor Cells. Cancer Immunol Res. 2017;5(1):3-8. 
doi:10.1158/2326-6066.CIR-16-0297 
8.  Manz MG, Boettcher S. Emergency granulopoiesis. Nat Rev Immunol. 2014;14(5):302-314. 
doi:10.1038/nri3660 
9.  Angka L, Martel AB, Kilgour M, et al. Natural Killer Cell IFNγ Secretion is Profoundly Suppressed 
Following Colorectal Cancer Surgery. Ann Surg Oncol. 2018;25(12):3747-3754. doi:10.1245/s10434-
018-6691-3 
10.  Zhang J, Tai LH, Ilkow CS, et al. Maraba MG1 virus enhances natural killer cell function via 
conventional dendritic cells to reduce postoperative metastatic disease. Mol Ther. 2014;22(7):1320-
1332. doi:10.1038/mt.2014.60 
11.  Mitroulis I, Ruppova K, Wang B, et al. Modulation of Myelopoiesis Progenitors Is an Integral 
Component of Trained Immunity. Cell. 2018;172(1-2):147-161.e12. doi:10.1016/j.cell.2017.11.034 
12.  Kaufmann E, Sanz J, Dunn JL, et al. BCG Educates Hematopoietic Stem Cells to Generate Protective 
Innate Immunity against Tuberculosis. Cell. 2018;172(1-2):176-190.e19. 
doi:10.1016/j.cell.2017.12.031 

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13.  Netea MG, Joosten LAB, van der Meer JWM. Hypothesis: stimulation of trained immunity as 
adjunctive immunotherapy in cancer. J Leukoc Biol. 2017;102(6):1323-1332. 
doi:10.1189/jlb.5RI0217-064RR 
14.  Bazett M, Costa AM, Bosiljcic M, et al. Harnessing innate lung anti-cancer effector functions with a 
novel bacterial-derived immunotherapy. Oncoimmunology. 2018;7(3):e1398875. 
doi:10.1080/2162402X.2017.1398875 
15.  Kalyan S, Bazett M, Sham HP, et al. Distinct inactivated bacterial-based immune modulators vary in 
their therapeutic efficacies for treating disease based on the organ site of pathology. Sci Rep. 
2020;10(1):5901. doi:10.1038/s41598-020-62735-z 
16.  Faraj TA, McLaughlin CL, Erridge C. Host defenses against metabolic endotoxaemia and their impact 
on lipopolysaccharide detection. Int Rev Immunol. 2017;36(3):125-144. 
doi:10.1080/08830185.2017.1280483 
17.  Sham HP, Bazett M, Bosiljcic M, et al. Immune Stimulation Using a Gut Microbe-Based 
Immunotherapy Reduces Disease Pathology and Improves Barrier Function in Ulcerative Colitis. 
Front Immunol. 2018;9:2211. doi:10.3389/fimmu.2018.02211 
18.  Sutcliffe S, Kalyan S, Pankovich J, et al. Novel Microbial-Based Immunotherapy Approach for Crohn’s 
Disease. Frontiers in Medicine. 2019;6:170. doi:10.3389/fmed.2019.00170 
19.  Hackl C, Neumann P, Gerken M, Loss M, Klinkhammer-Schalke M, Schlitt HJ. Treatment of colorectal 
liver metastases in Germany: a ten-year population-based analysis of 5772 cases of primary 
colorectal adenocarcinoma. BMC Cancer. 2014;14:810. doi:10.1186/1471-2407-14-810 
20.  Manfredi S, Lepage C, Hatem C, Coatmeur O, Faivre J, Bouvier AM. Epidemiology and Management 
of Liver Metastases From Colorectal Cancer. Ann Surg. 2006;244(2):254-259. 
doi:10.1097/01.sla.0000217629.94941.cf 
21.  Engstrand J, Nilsson H, Strömberg C, Jonas E, Freedman J. Colorectal cancer liver metastases – a 
population-based study on incidence, management and survival. BMC Cancer. 2018;18:78. 
doi:10.1186/s12885-017-3925-x 
22.  Nordlinger B, Sorbye H, Glimelius B, et al. Perioperative chemotherapy with FOLFOX4 and surgery 
versus surgery alone for resectable liver metastases from colorectal cancer (EORTC Intergroup trial 
40983): a randomised controlled trial. Lancet. 2008;371(9617):1007-1016. doi:10.1016/S0140-
6736(08)60455-9 
23.  Portier G, Elias D, Bouche O, et al. Multicenter randomized trial of adjuvant fluorouracil and folinic 
acid compared with surgery alone after resection of colorectal liver metastases: FFCD ACHBTH AURC 
9002 trial. J Clin Oncol. 2006;24(31):4976-4982. doi:10.1200/JCO.2006.06.8353 
24.  Kanemitsu Y, Shimizu Y, Mizusawa J, et al. Hepatectomy Followed by mFOLFOX6 Versus 
Hepatectomy Alone for Liver-Only Metastatic Colorectal Cancer (JCOG0603): A Phase II or III 

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Randomized Controlled Trial. J Clin Oncol. Published online September 14, 2021:JCO2101032. 
doi:10.1200/JCO.21.01032 
25.  Oxnard GR, Paweletz CP, Kuang Y, et al. Noninvasive detection of response and resistance in EGFR-
mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin 
Cancer Res. 2014;20(6):1698-1705. doi:10.1158/1078-0432.CCR-13-2482 
26.  Goldberg SB, Narayan A, Kole AJ, et al. Early Assessment of Lung Cancer Immunotherapy Response 
via Circulating Tumor DNA. Clin Cancer Res. 2018;24(8):1872-1880. doi:10.1158/1078-0432.CCR-17-
1341 
27.  Dindo D, Demartines N, Clavien PA. Classification of Surgical Complications: A New Proposal With 
Evaluation in a Cohort of 6336 Patients and Results of a Survey. Annals of Surgery. 2004;240(2):205-
213. doi:10.1097/01.sla.0000133083.54934.ae 
28.  Surgical Site Infection Event (SSI). National Healthcare Safety Network, Centers for Disease Control 
and Prevention; 2022. 
29.  Koch M, Garden OJ, Padbury R, et al. Bile leakage after hepatobiliary and pancreatic surgery: a 
definition and grading of severity by the International Study Group of Liver Surgery. Surgery. 
2011;149(5):680-688. doi:10.1016/j.surg.2010.12.002 
30.  Rahbari NN, Garden OJ, Padbury R, et al. Posthepatectomy liver failure: a definition and grading by 
the International Study Group of Liver Surgery (ISGLS). Surgery. 2011;149(5):713-724. 
doi:10.1016/j.surg.2010.10.001 
31.  Leslie K, Troedel S, Irwin K, et al. Quality of recovery from anesthesia in neurosurgical patients. 
Anesthesiology. 2003;99(5):1158-1165. doi:10.1097/00000542-200311000-00024 
32.  Myles PS, Weitkamp B, Jones K, Melick J, Hensen S. Validity and reliability of a postoperative quality 
of recovery score: the QoR-40. Br J Anaesth. 2000;84(1):11-15. 
doi:10.1093/oxfordjournals.bja.a013366 
33.  Shida D, Wakamatsu K, Tanaka Y, et al. The postoperative patient-reported quality of recovery in 
colorectal cancer patients under enhanced recovery after surgery using QoR-40. BMC Cancer. 
2015;15:799. doi:10.1186/s12885-015-1799-3 
34.  Peng LH, Wang WJ, Chen J, Jin JY, Min S, Qin PP. Implementation of the pre-operative rehabilitation 
recovery protocol and its effect on the quality of recovery after colorectal surgeries. Chin Med J 
(Engl). 2021;134(23):2865-2873. doi:10.1097/CM9.0000000000001709 
35.  Lee JH, Kim D, Seo D, Son JS, Kim DC. Validity and reliability of the Korean version of the Quality of 
Recovery-40 questionnaire. Korean J Anesthesiol. 2018;71(6):467-475. doi:10.4097/kja.d.18.27188 
36.  Tanaka Y, Wakita T, Fukuhara S, et al. Validation of the Japanese version of the quality of recovery 
score QoR-40. J Anesth. 2011;25(4):509-515. doi:10.1007/s00540-011-1151-2 

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37.  McMillan DC, Crozier JEM, Canna K, Angerson WJ, McArdle CS. Evaluation of an inflammation-based 
prognostic score (GPS) in patients undergoing resection for colon and rectal cancer. Int J Colorectal 
Dis. 2007;22(8):881-886. doi:10.1007/s00384-006-0259-6 
38.  Shrotriya S, Walsh D, Bennani-Baiti N, Thomas S, Lorton C. C-Reactive Protein Is an Important 
Biomarker for Prognosis Tumor Recurrence and Treatment Response in Adult Solid Tumors: A 
Systematic Review. PLoS One. 2015;10(12):e0143080. doi:10.1371/journal.pone.0143080 
39.  Frühling P, Hellberg K, Ejder P, Strömberg C, Urdzik J, Isaksson B. The prognostic value of C-reactive 
protein and albumin in patients undergoing resection of colorectal liver metastases. A retrospective 
cohort study. HPB. 2021;23(6):970-978. doi:10.1016/j.hpb.2020.10.019 
40.  Punt CJA, Buyse M, Köhne CH, et al. Endpoints in adjuvant treatment trials: a systematic review of 
the literature in colon cancer and proposed definitions for future trials. J Natl Cancer Inst. 
2007;99(13):998-1003. doi:10.1093/jnci/djm024 
41.  Karanicolas PJ, Farrokhyar F, Bhandari M. Blinding: Who, what, when, why, how? Can J Surg. 
2010;53(5):345-348. 
42.  Marshall JC. The Multiple Organ Dysfunction Syndrome. Zuckschwerdt; 2001. Accessed April 22, 
2022. http://www.ncbi.nlm.nih.gov/books/NBK6868/ 
43.  Bone RC, Balk RA, Cerra FB, et al. Definitions for Sepsis and Organ Failure and Guidelines for the Use 
of Innovative Therapies in Sepsis. Chest. 1992;101(6):1644-1655. doi:10.1378/chest.101.6.1644 
44.  Tsai MS, Su YH, Ho MC, et al. Clinicopathological Features and Prognosis in Resectable Synchronous 
and Metachronous Colorectal Liver Metastasis. Ann Surg Oncol. 2007;14(2):786-794. 
doi:10.1245/s10434-006-9215-5 
45.  Lochan R, White SA, Manas DM. Liver resection for colorectal liver metastasis. Surgical Oncology. 
2007;16(1):33-45. doi:10.1016/j.suronc.2007.04.010 
46.  Bismuth H, Chiche L. Surgery of hepatic tumors. Prog Liver Dis. 1993;11:269-285. 
47.  Soules MR, Sherman S, Parrott E, et al. Executive summary: Stages of Reproductive Aging Workshop 
(STRAW). Climacteric. 2001;4(4):267-272. 
 

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APPENDIX A: CIOMS Form 
 

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APPENDIX B: Contraceptive Guidance 
Participants of Childbearing Potential: 
Women of childbearing potential (WOCBP) are participants of female sex who do not meet either of the 
following criteria: 
1. Postmenopausal participants. Menopause will be clinically defined as occurring 12 months after 
a participant’s last menstrual period.47 An elevated follicle stimulating hormone level in the 
postmenopausal range may be used to confirm a postmenopausal state in women not using 
hormonal contraception or hormonal replacement therapy. However, in the absence of 12 
months of amenorrhea, follicle stimulating hormone levels will be insufficient to confirm a 
postmenopausal state. Women on hormone replacement therapy or whose menopausal status 
remains unknown will be assumed to be WOCBP and required to use one of the non-estrogen 
containing highly effective contraception methods listed in the subsequent section titled 
“Contraception Guidance”. 
2. Participants with a prior hysterectomy, bilateral oophorectomy, and/or bilateral salpingectomy. 
Documentation of these procedures can come from a study personnel’s review of medical 
records, imaging, or medical history interview. 
 
Fertile men are participants of male sex who have not undergone a vasectomy (without subsequent 
reversal) or bilateral orchiectomy. 
Contraception Guidance: 
 
Participants who are WOCBP or fertile men participating in penile-vaginal intercourse must practice at 
least 1 highly effective method of contraception with their partners from the time of signing the ICF 
through 2 months after the last dose of IP, OR these participants must agree to completely abstain from 
penile-vaginal intercourse. Acceptable methods of contraception are listed in Table 2. 
 
Fertile men must also refrain from donating sperm while receiving IP  
 
All participants of male sex with a pregnant or breastfeeding partner must agree to use a male condom 
or remain abstinent from penile-vaginal intercourse while receiving IP  
 
Table 2. Highly Effective Contraceptive Methods 
User Dependent Methodsa 
  Combined (estrogen and progesterone containing) hormonal contraception associated 
with inhibition of ovulation 
  - Oral 
  - Intravaginal 

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  - Transdermal 
  Progesterone-only hormonal contraception associated with inhibition of ovulation 
  - Oral 
  - Injectable 
  Male Condom 
  Abstinence 
  - Considered highly effective only if participant is completely compliant. The reliability 
needs to be evaluated in relation to the duration of treatment with IP and the 
preferred and usual lifestyle of the participant 
User Independent Methods 
  Implantable progesterone-only hormonal contraception associated with inhibition of 
ovulation 
  Progesterone or Copper IUD 
  Bilateral tubal occlusion or ligation 
  Vasectomy 
  - Considered highly effective provided that the absence of sperm has been confirmed 
IUD: intrauterine device 
a Typical use failure rates may differ from those when used consistently and correctly.  
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX C: Summary of IP Procedures, Study Specific Tests, and Analysis Cohorts According to Clinical 
Scenario 
 
Participant 
IP 
Study Specific Tests 
Primary 
Analysis 
Correlative 
Analysis 
Sensitivity 
Analysis 
Safety 
Analysis 
1 
Undergoes surgery with 
resection +/- ablation of 
all CRLMs and received 
a minimum of 80% of 
the prescribed 
preoperative doses of IP 
and at least 90% of the 
postoperative doses. 
Continue to 
end 
PFS/OS: as scheduled 
ctDNA: as scheduled 
Side effect profile: as 
scheduled 
QoR: as scheduled 
Correlative outcomes: as 
scheduled 
 
Yes 
Yes 
Yes 
Yes 
2 
Stops IP due to toxicity, 
patient preference, 
clinician preference, or 
MODS and received less 
than 80% of the 
prescribed preoperative 
doses of IP or less than 
90% of the 
postoperative doses. 
Stop 
PFS/OS: as scheduled 
ctDNA: as scheduled 
Side effect profile: as 
scheduled 
QoR: as scheduled 
Correlative outcomes: as 
scheduled 
 
Yes 
Yes 
No 
Yes 
3 
Does not undergo 
surgery due to 
progression, the patient 
is too unwell, patient 
preference, or >120 
days have elapsed since 
starting IP for reasons 
other than apparent 
cCR or withdrawal. 
Stop at the 
time of the 
decision to 
not undergo 
surgery, or 
after 120 
days if 
sooner 
PFS/OS: as scheduled 
ctDNA: measured once 
after the decision to not 
operate (within 14 days 
of the decision) 
Side effect profile: as 
scheduled 
QoR: stop 
Correlative outcomes: 
stop 
Yes 
No 
No 
Yes 
4 
Does not have surgery 
due to apparent cCR 
Continue x 
additional 41 
days from 
time of 
decision not 
to operate 
PFS/OS: all assessments 
with timing defined 
based on decision not to 
operate 
ctDNA: all assessments 
with timing defined 
based on decision not to 
operate 
Side effect profile: all 
assessments with timing 
defined based on 
decision not to operate  
QoR: stop 
Correlative outcomes: 
stop 
Yes 
No 
Yes 
Yes 
5 
Has surgery but no 
resection or ablation 
due to apparent cCR 
Continue to 
end 
PFS/OS: as scheduled 
ctDNA: as scheduled 
Yes 
Yes 
Yes 
Yes 

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Side effect profile: as 
scheduled 
QoR: as scheduled 
Correlative outcomes: as 
scheduled 
6 
Has surgery but not a 
complete resection or 
ablation due to 
progression, 
unresectable disease, or 
any other reason 
excluding apparent cCR  
Stop at the 
time of 
surgery 
PFS/OS: as scheduled 
ctDNA: measured once 
postoperatively 
Side effect profile: as 
scheduled 
QoR: as scheduled 
Correlative outcomes: 
All pre-operative 
measurements. 
Postoperative 
measurements only on 
POD1, POD4 (+/- 1 day), 
and post-op 6 weeks (+/- 
10 days) 
Yes 
Yes 
No 
Yes 
7 
Has surgery with the 
resection of 
extrahepatic 
metastases discovered 
at the time of the 
operation 
Continue to 
end 
PFS/OS: as scheduled 
ctDNA: as scheduled 
Side effect profile: as 
scheduled 
QoR: as scheduled 
Correlative outcomes: as 
scheduled 
Yes – and 
do not 
count as an 
event 
Yes 
No 
Yes 
8 
Has surgery with 
resection or ablation of 
all known disease; 
however, after surgery 
found to have 
extrahepatic disease 
that was present prior 
to surgery 
Stop when 
progression 
is 
determined  
PFS/OS: as scheduled 
ctDNA: as scheduled 
Side effect profile: as 
scheduled 
QoR: as scheduled 
Correlative outcomes: as 
scheduled 
Yes – PFS 
defined as 
the date of 
progression 
Yes 
No 
Yes 
9 
Has surgery with 
resection or ablation, 
and surgical pathology 
is not CRLM (and not 
cCR) 
Stop when 
the 
pathology is 
reviewed by 
investigators 
PFS/OS: stop 
ctDNA: stop 
Side effect profile: as 
scheduled 
QoR: as scheduled 
Correlative outcomes: 
stop 
No 
Yes 
No 
Yes 
10 
Chooses to withdraw 
*If participant withdraws 
from the study before taking 
any IP, there will be no 
further study specific tests 
and will not be part of any 
analysis. They will also be 
replaced with another 
participant  
Stop at time 
of 
withdrawal 
If willing: 
PFS/OS: as scheduled 
ctDNA: as scheduled 
Side effect profile: as 
scheduled 
QoR: as scheduled 
Correlative outcomes: as 
scheduled 
If willing 
If willing 
No 
Yes 
IP: investigational product, PFS: progression free survival, OS: overall survival, ctDNA: circulating tumour DNA, QoR: quality of 
recovery, cCR: clinical complete response. 

 ____________________________________________________________________________________________ 
Protocol Number: 4023 
Version: 4.0  dated 19.AUG.2024 
49