The x- axis shows the number of positive tests, and the y -axis shows the probabilities. Figure S1. Distribution of the Expected Number of Positive Tests Based on the simulation, at lea st 7 subjects are expected to have an immune response against 2 or more cancer antigens with at least 97.5% probability. The expected distribution of probabilities having a given number of subjects with positive immune response for 2 or more antigens is di splayed in Figure S2, the x-axis shows the number of subjects with a given probability and the y- axis shows the probabilit y. Figures S2. Expected Distribution of Probabilities Having a Given Number of Subjects with Positive Immu ne Response for Two or More Antigens Statistical Methods and Analyses: Demographic and baseline characteristics will be summarized using descriptive statistics. Safety/tolerability will be assessed by tabulating the number and percentage of subjects who develop 1 or more related Grade 3 or higher systemic SAEs and/or Grade 4 or higher local SAEs. The incidence of AEs, the incidence of treatment -related AEs, and the severity of AEs will be summarized using descriptive statistics. Other safety parameters (injection site reactions, clinical laboratory parameters, vital signs, ECG measures) and the cha nge from Baseline in these parameters will be summarized using descriptive statistics. 00.020.040.060.08 0 20 40 60 80 00.10.20.30.40.5 0 5 10 Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 10 of 81 CONFIDENTIAL Immunogenicity parameters versus time will be plotted for each subject; similar summary plots will be constructed for the 10 subjects. Efficacy parameters versus time will be plotted for each subject; similar summary plots will be constructed for the 10 subjects. Correlation and significance tests will be performed on the results of exploratory measures . Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 11 of 81 CONFIDENTIAL TABLE OF CONTENTS Page CLINICAL STUDY PROTOCOL .........................................................................................1 CLINICAL STUDY PROTOCOL APPROVAL SIGNATURE PAGE .............................2 INVESTIGATOR ACCE PTANCE PAGE ...........................................................................3 SYNOPSIS ................................................................................................................................4 TABLE OF CONTENTS ......................................................................................................11 LIST OF TABLES .................................................................................................................15 LIST OF FIGURES ...............................................................................................................15 LIST OF ABBREVIATION S AND DEFINITIONS OF TERMS ....................................16 1 BACKGROUND INFORMATI ON .........................................................................18 1.1 Colorectal Cancer Background ........................................................................18 1.2 PolyPEPI1018 and Companion Diagnostic Vaccine Overview ......................19 1.2.2 Preclinical and Clinical Background for PolyPEPI1018 ..................22 1.3 Rationale for Study Design and Dose Regimen ..............................................24 1.4 Benefits and Risks............................................................................................26 2 STUDY OBJECTIVES ..............................................................................................28 2.1 Primary Objectives ...........................................................................................28 2.2 Secondary Objectives .......................................................................................28 2.3 Exploratory Objectives ....................................................................................28 3 STUDY DESIGN ........................................................................................................29 3.1 Overall Study Design .......................................................................................29 3.2 Outcome Measures ...........................................................................................31 3.2.1 Primary Outcome Measures ..............................................................31 3.2.2 Secondary Outcome Measures ..........................................................31 3.2.3 Exploratory Outcome Measures .......................................................32 3.3 Estimated Study Duration ................................................................................32 3.4 Randomization and Blinding ...........................................................................33 3.5 Study Stopping Rules .......................................................................................33 4 SUBJECT SELECTION ...........................................................................................34 4.1 Inclusion Criteria .............................................................................................34 Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 12 of 81 CONFIDENTIAL 4.2 Exclusion Criteria ............................................................................................35 4.3 Subject Withdrawal and Discontinuation ........................................................36 4.3.1 Permanent Treatment Discontinuation .............................................38 4.3.2 Premature Treatment Discontinuation ..............................................38 5 STUDY TREATMENTS ...........................................................................................39 5.1 PolyPEPI1018 CRC Vaccine ...........................................................................39 5.1.1 Description ........................................................................................39 5.1.2 PolyPEPI1018 Product Preparation and Administration ..................39 5.1.3 Packaging and Labeling ....................................................................40 5.1.4 Supply, Distribution, and Pharmacy .................................................40 5.1.5 Storage ..............................................................................................41 5.2 Reference Therapy ...........................................................................................41 5.3 Investigational Product Accountability ............................................................41 5.4 Concomitant Medications ................................................................................42 5.4.1 Required Medications .......................................................................42 5.4.2 Prohibited Medications .....................................................................42 5.4.3 Precautionary Medications ................................................................43 5.5 Dose Modification Guidelines and Toxicity Management ..............................43 5.5.1 Local Reactions .................................................................................43 5.5.2 Systemic Reactions ...........................................................................44 5.5.3 Anticancer Drugs ..............................................................................44 5.5.4 Dose Limiting Toxicity (DLT) .........................................................45 6 DESCRIPTION OF STUDY ASSESSMENTS AND PRO CEDURES .................46 6.1 Informed Consent Form ...................................................................................46 6.2 Subject Identification Code ..............................................................................46 6.3 Screening Log ..................................................................................................46 6.4 Vaccine Site Evaluation ...................................................................................47 6.5 Evaluation of Response to First- line Therapy .................................................47 6.6 Immunogenicity and Efficacy Asses sments ....................................................47 6.6.1 PEPI Test ..........................................................................................48 6.6.2 Effector T Cell Response ..................................................................48 6.6.3 Memory T Cell Response .................................................................48 6.6.4 Tumor Infiltrating Lymphocyte (TILs) .............................................49 Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 13 of 81 CONFIDENTIAL 6.6.5 CT scan and RECIST/ irRECIST evaluation ....................................49 6.7 Safety Assessments ..........................................................................................50 6.7.1 Adverse Events .................................................................................50 6.7.2 Urgent Safety Measures ....................................................................50 6.7.3 Untoward Medical Occurrences .......................................................50 6.7.4 Non-Medical Complaints ..................................................................51 6.7.5 Medical, Medication, and Non- Drug Therapy History .....................51 6.7.6 Physical Examinations ......................................................................52 6.7.7 Vital Signs .........................................................................................52 6.7.8 Clinical Laboratory Parameters ........................................................53 6.7.9 Patient Diary .....................................................................................54 6.8 Procedures for Monitoring Subject Compliance..............................................54 7 ASSESSMENTS AND PROCEDURES BY STUDY VISIT ..................................55 7.1 Screening Period (Visit 1 - Days -21 to Day -1 – Weeks -3 to -1) ..................55 7.2 PolyPEPI1018 Administration (Visit 2 – Day 1 - Week 0), (Visit 7 – Day 92 ±2 - Week 13) and (Visit 12 – Day 183 ±2 - Week 26), .....................55 7.3 Follow-Up Period.............................................................................................56 7.3.1 Visit 3 (Day 22 - Week 3), Visit 5 (Day 64 - Week 9), Visit 8 (Day 113 ±2 - Week 16), Visit 10 (Day 155 ±2 - Week 22), Visit 13 (Day 204 ±2 - Week 29) and Visit 15 (Day 246 ±2 - Week 35) ...........................................................................................56 7.3.2 Visit 4 (Day 43 - Week 6), Visit 6 (Day 85 - Week 12), Visit 9 (Day 134 ±2 - Week 19), Visit 11 (Day 176 ±2 - Week 25), Visit 14 (Day 225 ±2 - Week 32) and Visit 16 (Day 267 ±2 - Week 38) ...........................................................................................56 7.4 Last Visit ..........................................................................................................57 8 ADVERSE EVENTS AND SERIOUS ADVERSE EVENT S ................................58 8.1 Definitions........................................................................................................58 8.1.1 Adverse Event ...................................................................................58 8.1.2 Serious Adverse Event ......................................................................58 8.1.3 Suspected Unexpected Serious Adverse Reaction (SUSAR) ...........59 8.1.4 Non-Serious Adverse Event ..............................................................59 8.1.5 Unexpected Adverse Events .............................................................59 8.1.6 Preexisting Diseases ..........................................................................59 8.2 Assessment of Adverse Events ........................................................................60 Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 14 of 81 CONFIDENTIAL 8.2.1 Severity .............................................................................................61 8.2.2 Causality ...........................................................................................61 9 STATISTICS ..............................................................................................................63 9.1 Analysis Populations ........................................................................................63 9.2 Replacement of Subjects ..................................................................................63 9.3 Handling of Missing, Unused, and Spurious Data ...........................................63 9.3.1 Missing Data Resulting from Study Withdrawal ..............................63 9.3.2 Intermediate Missing Data ................................................................63 9.4 Primary Endpoint .............................................................................................63 9.5 Statistical Analyses ..........................................................................................64 9.5.1 Safety Analysis .................................................................................64 9.5.2 PEPI and Immunogenicity Analyses ................................................65 9.5.3 Efficacy Analyses .............................................................................65 9.5.4 Exploratory Analyses ........................................................................66 9.6 Determination of Sample Size .........................................................................66 10 ETHICAL AND LEGAL CONSIDERATIONS .....................................................69 10.1 Compliance Statement .....................................................................................69 10.2 Ethics Committee and Regulatory Authorities ................................................69 10.3 Informed Consent and Human Subject Protection...........................................69 10.4 Confidentiality .................................................................................................70 11 ADMINISTRATIVE CONSIDERATION ..............................................................71 11.1 Investigator’s Responsibilities .........................................................................71 11.2 Study Training .................................................................................................71 11.3 Study Monitoring .............................................................................................71 11.4 Safety Monitoring ............................................................................................72 11.5 Audits ...............................................................................................................72 11.6 Documentation .................................................................................................72 11.6.1 C ase Report Forms and Study Records ............................................72 11.6.2 Direct Access to Source Data, Source Documents, and Study Records .............................................................................................73 11.6.3 Source Data .......................................................................................73 11.6.4 Document and Data Retention ..........................................................74 11.7 Non-Compliance with the Protocol .................................................................74 Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 15 of 81 CONFIDENTIAL 11.8 Financing and Insurance ..................................................................................75 11.9 Publication Policy ............................................................................................75 12 REFERENCES ...........................................................................................................76 APPENDIX A. SCHEDULE OF STUDY AS SESSMENTS ..................................78 APPENDIX B.