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| CLINICAL STUDY PROTOCOL   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
|---------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Protocol Title:           | A Phase 1, Multiple doses, Multicenter, Open-label Study to  Evaluate Safety, Tolerability, Immunogenicity and Efficacy of  Subcutaneous Injections of PolyPEPI1018 Vaccine as an Add-on  Immunotherapy to the Standard-of-Care Maintenance Therapy in  Subjects with Metastatic Colorectal Cancer (OBERTO)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| Short Protocol Title:     | Safety and Immunogenicity of PolyPEPI1018 Vaccine in the  Treatment of Metastatic Colorectal Cancer (OBERTO)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| Protocol Number:          | OBERTO-101 Amendment 3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| Name of Investigational   | PolyPEPI1018 CRC Vaccine (PolyPEPI1018)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |
| Product: IND Number:      | 17887 #0001                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| EudraCT Number:           | 2017-003850-18                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| Sponsor:                  | Treos Bio ZRT Viola utca, 2 8200 Veszprém  Hungary                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| Study Medical Monitor:    | Franco Lori, M.D., Ph.D. Chief Medical Officer at Treos Bio ZRT Phone: +39 347 0723942  FAX: +36-1-332-3636  E-mail: franco.lori@treosbio.com                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| Original Protocol Date:   | 15 November 2017                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| Amendment 1 Date          | 15 January 2018                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| Amendment 2 Date          | August 21 2018                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
| Amendment 3 Date          | April 10 2019                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |
| Confidentiality           | This document contains information that is the confidential and proprietary  property of Treos Bio ZRT. Any use, distribution, or disclosure without the                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
| Statement:                | prior written consent of Treos Bio ZRT is strictly prohibited except to the  extent required under applicable laws or regulations.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| Compliance Statement:     | This study will be conducted in accordance with Good Clinical Practice (GCP)  as defined in International Conference on Harmonisation (ICH) guidelines and  United States (US) Code of Federal Regulations Title 21, Parts 11, 50, 54, 56,  and 312, and Title 45 Parts 46, 160, and 164; the International Council for  Harmonisation (ICH) document ICH Harmonised Guideline, E6(R2) Integrated  Addendum to ICH E6(R1):Guideline for Good Clinical Practice; the European  Union (EU) Directives 2001/20/EC and 2005/28/EC; the Declaration of  Helsinki (version as currently endorsed by the European Medicines Agency  [EMA] and the US Food and Drug Administration [FDA], 1989); Institutional  Review Board/Independent Ethics Committee (IRB/IEC) Guidelines; and  applicable local legal and regulatory requirements. |
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clinical study protocol approval signature

| Protocol Title:                                 | A Phase 1, Multiple doses, Multicenter, Open-label Study to  Evaluate Safety, Tolerability, Immunogenicity and Efficacy of  Subcutaneous Injections of PolyPEPI1018 Vaccine as an Addon Immunotherapy to the Standard-of-Care Maintenance  Therapy in Subjects with Metastatic Colorectal Cancer (OBERTO)                             |
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| Protocol Number:                                | OBERTO-101 15 November 2017 |
| Date of Original  Protocol: Date of Amendment 1 | 15 January 2018             |
| Date of Amendment 2                             | August 21 2018              |
| Date of Amendment 3                             | April 10 2018               |  
APPROVAL STATEMENT The undersigned has reviewed the format and content of the above protocol and approved for issuance. Approved by:
Franco Lori, M.D., Ph.D.  
Chief Medical Officer 10th April 2019 INVESTIGATOR **ACCEPTANCE PAGE**
Issue/Date: 15 November 2017 Date of Amendment 1: 15 January 2018 Date of Amendment 2: 21 August 2018 Date of Amendment 3: 10 April 2019 I have read this protocol for Study OBERTO-101 entitled: A Phase 1, Multiple doses, Multicenter, Open-label Study to Evaluate Safety, Tolerability, Immunogenicity and Efficacy of Subcutaneous Injections of PolyPEPI1018 Vaccine as an Add-on Immunotherapy to the Standard-of-Care Maintenance Therapy in Subjects with Metastatic Colorectal Cancer (OBERTO)
By signing below, the investigator acknowledges that he/she has read and understands this protocol, and will comply with the requirements for obtaining informed consent from all study subjects prior to initiating any protocol-specific procedures, obtaining written initial and ongoing Ethics Committees protocol review and approval, understands and abides by the requirements for maintenance of source documentation, and provides assurance that this study will be conducted according to all requirements as defined in this protocol, Clinical Trial Agreement, ICH GCP guidelines, and all applicable national and local regulatory requirements.  
Investigator Name (print) Investigator Signature Date  
| SYNOPSIS                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |
|-----------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Name of                           | Treos Bio ZRT                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |
| Sponsor/Company: Name of Product: | PolyPEPI1018 CRC Vaccine (PolyPEPI1018)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| Title of Study:                   | A Phase 1, Multiple doses, Multicenter, Open-label Study to Evaluate Safety,  Tolerability, Immunogenicity and Efficacy of Subcutaneous Injections of  PolyPEPI1018 Vaccine as an Add-on Immunotherapy to the Standard-ofCare Maintenance Therapy in Subjects with Metastatic Colorectal Cancer (OBERTO)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |
| Phase of Development:             | Phase 1; First-in-Human                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| Planned Study Period:             | Initiation: Second quarter of 2018 (first subject first visit)  Primary Completion: Third quarter of 2019 (last subject Week 12  assessment)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
| Study Objective(s):               | Primary Objective: - To evaluate the safety and tolerability of multiple doses of  PolyPEPI1018 as an add-on to maintenance therapy in subjects with  metastatic colorectal cancer (mCRC) Secondary Objective(s):  - To identify PEPIs (Personal EPItopes capable of inducing T cell  responses in an individual) from PolyPEPI1018 in each study subject - To evaluate the immunogenicity of PolyPEPI1018 by measuring both  effector and memory T cell responses  - To evaluate initial efficacy of PolyPEPI1018 by evaluating Objective  Response Rate (ORR)  Exploratory Objective(s):  - To explore the correlation between PEPIs and T cell responses  - To explore the correlation between PEPIs and tumor infiltrating  lymphocytes  - To explore the correlation between T cell responses and tumor  infiltrating lymphocytes  - To explore the correlation between PEPIs and ORR  - To explore the correlation between T cell responses and ORR                                                                                                |
| Study Design:                     | This is a Phase 1, open-label, non-randomized, multicenter study to evaluate  the safety, tolerability, immunogenicity and efficacy of multiple subcutaneous (SC) injections of PolyPEPI1018 as an add-on immunotherapy  to the standard-of-care maintenance therapy in approximately 10 subjects  with mCRC. This is an amended Study Design only in the USA: 5 subjects in  Italy are under evaluation after a single SC injection of the same vaccine, as  originally planned (no amendment contemplated in Italy).  The study is composed of a 3-week Screening Period, the administration of  multiple doses vaccine (Day 1, Week 0; Day 92, Week 13; Day 183, week  26), a 13-week Follow-up Period between the first and second vaccination  and a 12-week Follow-up Period after the last vaccination. The study will be  conducted on an outpatient basis. Screening should be performed in parallel with the subject's completion of  the standard-of-care first-line treatment and initiation of the standard-of-care  maintenance treatment. |  
| Treos Bio ZRT                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | OBERTO-101 Amendment 3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                    |
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| Product: PolyPEPI1018 Colorectal Cancer Vaccine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Page 5 of 81                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                    |
| The first dose of PolyPEPI1018 will be administered after the subject  initiates the maintenance regimen, and within 3 weeks after the eligibility CT scan was performed (should be performed at the completion of the first-line  treatment).  Subjects will be monitored every 3 weeks for 12 weeks after first  administration of PolyPEPI1018 (i.e. Weeks 3, 6, 9 and 12), then vaccinated  again at week 13 and monitored every 3 weeks for 12 weeks after second  administration of PolyPEPI1018 (i.e. Weeks 16, 19, 22 and 25), then  vaccinated again at week 26 and monitored every 3 weeks for 12 weeks after  third administration of PolyPEPI1018 (i.e. Weeks 29, 32, 35 and 38) |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |                                                                                                                                                    |
| Number of Investigational                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Two sites, 1 in the United States (US) and 1 in Europe (Italy)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |                                                                                                                                                    |
| Sites: Planned Number of                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Approximately 10 subjects in the USA, to be vaccinated 3 times (USA), in                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                    |
| Subjects:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | addition to 5 subjects to be vaccinate only 1 time (Italy)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                    |
| Planned Duration of                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Approximately 41 weeks, including a 3-week Screening Period and 12-week                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                    |
| Subject Participation:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | Follow-up Period after third and last PolyPEPI1018 dose is administered                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                    |
| Study Population:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | Patients with mCRC who have achieved partial response (PR) or stable  disease during first-line treatment with chemotherapy plus a biologic drug will be enrolled                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                    |
| Test Product, Dose, and                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | The final dosage form of PolyPEPI1018 is an emulsified solution prepared                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                    |
| Mode of Administration:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | onsite, by mixing 2 separate solutions of multiple peptides  (PolyPEPI1018-Mix1 and PolyPEPI1018-Mix2) and Montanide™ as an  adjuvant.  The sponsor will provide the following:  - PolyPEPI1018-Mix1 - a solution of 2 × 30-mer peptides at  0.2 mg/peptide/mL in 1.5 mL solution (20% DMSO, 80% water) and  stored at -20ºC - PolyPEPI1018-Mix2 - a solution of 4 × 30-mer peptides at  0.2 mg/peptide/mL in 1.5 mL solution (20% DMSO, 80% water) and  stored at -20ºC The site staff will follow the written vaccine preparation procedure and mix  1.2 mL of PolyPEPI1018-Mix1, 1.2 mL of PolyPEPI1018-Mix2, and 2.4 mL of Montanide™ to produce the emulsified vaccine solution. After preparing  the mixture, 1 mL of the mixture must be injected SC into each of 4 selected  injection sites (1 mL SC injection per site). After the injections are  administered, the subject should be observed for at least 1 hour for any local  and/or systemic allergic reactions.  Dosage Form: Emulsified solution consisting of PolyPEPI1018-Mix1,  PolyPEPI1018-Mix2, and Montanide™ (adjuvant), prepared onsite and used  immediately post preparation.  Dose Level: 0.2 mg/ peptide, 6 peptides total Mode of Administration: SC injection |                                                                                                                                                    |
| Reference Therapy, Dose,  and Mode of  Administration:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | No reference therapy is used in this study                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                    |
| Inclusion Criteria:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | 1.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | Male or female subjects, 18-75 years of age at time of Screening who  provide written informed consent prior to initiation of any study  procedure |
| 2.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | Histologically confirmed metastatic adenocarcinoma originating from  the colon or the rectum                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                    |  
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| Product: PolyPEPI1018 Colorectal Cancer Vaccine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Page 6 of 81                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                          |
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| 3.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Presence of at least 1 measurable reference lesion according to the  Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 criteria                                                                                                                                                                                                                                                   |                                                                                                                                                          |
| 4.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Experienced PR or stable disease during first-line treatment with a  systemic chemotherapy regimen and 1 biological therapy regimen                                                                                                                                                                                                                                                               |                                                                                                                                                          |
| 5.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Maintenance therapy with a fluoropyrimidine (5-fluorouracil or  capecitabine) plus the same biologic agent (bevacizumab, cetuximab or  panitumumab) used during induction, scheduled to initiate prior to the  first day of treatment with the study drug                                                                                                                                         |                                                                                                                                                          |
| 6.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | No more than 1 line of chemotherapy regimen for mCRC (adjuvant  therapy for non-metastasized disease is allowed if terminated more than  6 months before Screening and without recurrence within 6 months after  the end of adjuvant treatment)                                                                                                                                                   |                                                                                                                                                          |
| 7.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Last CT scan at 3 weeks or less before the first day of treatment                                                                                                                                                                                                                                                                                                                                 |                                                                                                                                                          |
| 8.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Eastern Cooperative Oncology Group (ECOG) performance status of 0  or 1                                                                                                                                                                                                                                                                                                                           |                                                                                                                                                          |
| 9.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Women of childbearing potential must agree to appropriately use an  effective form of contraception (failure rate of <1% per year) for  3 months from the day of the treatment. An effective form of  contraception is defined as using hormonal contraceptives or an  intrauterine device combined with at least 1 of the following forms of  contraception: a diaphragm, cervical cap or condom |                                                                                                                                                          |
| 10. Men must agree to use an effective form of contraception (as defined  above), and not donate sperm for 3 months from the day of the treatment 11. White blood cell count ≥3.0 × 109 /L with neutrophils ≥1.5 × 109 /L  12. Platelets ≥100 × 109 /L, hemoglobin ≥5.6 mmol/L (corresponding to 9 g/dL) 13. Serum bilirubin ≤1.5 × upper limit of normal (ULN) set by the site 14. Alanine amino transferase (ALAT) and aspartate amino transferase (ASAT) ≤2.5 × ULN in the absence of liver metastases. ALAT and  ASAT ≤5 × ULN set by the site in the presence of liver metastases 15. Serum creatinine ≤1.5 × ULN set by the site and creatinine clearance  >30 mL/min using Cockroft formula 16. Relevant toxicities of prior therapies must have resolved to ≤Grade 1,  except for oxaliplatin-related neuropathy or alopecia 17. Anticipated life expectancy ≥6 months  18. Subject is willing and able to comply with the requirements of the  protocol |                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                          |
| Exclusion Criteria:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | 1.                                                                                                                                                                                                                                                                                                                                                                                                | Received chronic systemic immune therapy or immunosuppressant  medication other than steroids within the last 6 weeks prior to start of  study treatment |
| 2.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Received continuous systemic steroid treatment within the last 2 weeks  prior to start of study treatment                                                                                                                                                                                                                                                                                         |                                                                                                                                                          |
| 3.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Colorectal cancer with documented high microsatellite instability  (MSI-H)                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                          |
| 4.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Colorectal cancer with documented BRAF mutations                                                                                                                                                                                                                                                                                                                                                  |                                                                                                                                                          |
| 5.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Pre-existing systemic autoimmune or antibody-mediated diseases or  immune deficiency diseases                                                                                                                                                                                                                                                                                                     |                                                                                                                                                          |
| 6.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Central nervous system (CNS) metastases                                                                                                                                                                                                                                                                                                                                                           |                                                                                                                                                          |
| 7.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Active or uncontrolled severe infections or undiagnosed febrile  condition >38ºC                                                                                                                                                                                                                                                                                                                  |                                                                                                                                                          |  
Page 7 of 81  
| Product: PolyPEPI1018 Colorectal Cancer Vaccine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Page 7 of 81                                                                                                                                                                                                                                                                                                |
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| 8.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Acute or subacute intestinal obstruction or history of chronic intestinal  inflammatory diseases                                                                                                                                                                                                            |
| 9.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Symptomatic peritoneal carcinomatosis                                                                                                                                                                                                                                                                       |
| 10. Peritonitis  11. Serious, non-healing wounds, ulcers or bone fractures 12. Nephrotic syndrome  13. Arterial thromboembolisms or severe hemorrhages within 6 months  before study enrolment (except bleeding tumor before tumor resection  surgery)  14. Hemorrhagic diathesis or thrombotic tendency  15. Major surgery or radiotherapy within 12 weeks prior to the study  treatment or anticipation of needing such procedure during the study  period 16. Uncontrolled pleural effusion, pericardial effusion or ascites requiring  repeated drainage more than once every 28 days 17. Participants with active malignancy (other than colorectal cancer [CRC])  or a prior malignancy within the past 12 months 18. Participant with myocardial infarction within 6 months prior to  enrollment or New York Heart Association Class III or IV heart failure,  uncontrolled angina, severe uncontrolled ventricular arrhythmias or  electrocardiographic evidence of acute ischemia or active conduction  system abnormalities. Prior to the first dose of study treatment, any  electrocardiogram (ECG) abnormality at Screening must be documented  by the investigator as not medically relevant 19. Administration of a live, attenuated vaccine within 4 weeks before  randomization or anticipation of a live attenuated vaccine will be  required during the study 20. Participant has participated in another clinical study involving an  investigational product (IP) or investigational device within 30 days  prior to enrollment or is scheduled to participate in another clinical study  involving an IP or investigational device during the course of this study 21. Known hypersensitivity to any component of the investigational drug  22. If female, participant is pregnant (exclusion confirmed with beta-human  chorionic gonadotropin [hCG] test) or lactating at the time of enrollment,  or has plans to become pregnant or start breastfeeding during the study 23. Pre-existing alcohol or drug abuse  24. Medical or mental impairments which make it impossible to obtain the  patient's consent or to conduct the study 25. A significant concomitant medical condition which the clinical  investigator believes precludes the patient from enrolling in the study  26. Absent or limited legal competence |                                                                                                                                                                                                                                                                                                             |
| Primary Endpoint:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Occurrence of at least 1 ≥Grade 4 local adverse event (AE) or 1 ≥Grade 3  systemic AE and/or signs/symptoms, lab toxicities, and/or clinical events that  is probably or definitely related to study treatment, any time from Day 1 until  21 days after administration of each dose of the vaccine.        |
| Criteria for  Evaluation/Outcome  Measures:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Primary Outcome Measures: - The incidence and severity of all adverse events (AEs), related AEs, all  serious adverse events (SAEs), related SAEs, and temporally-associated  AEs according to the National Cancer Institute-Common Terminology  Criteria for Adverse Events (NCI-CTCAE) version 4.0 (v4.0) |  
- Change from baseline in clinical laboratory safety parameters and vital  
signs  
- Number and proportion of subjects with any clinically significant  
change in vital signs (i.e., blood pressure, pulse rate, respiratory rate,  
body temperature) during the vaccine administration or within  
60 minutes following administration  
Secondary Outcome Measure(s):  
- PEPIs as identified by the PEPI Test  
- Effector T cell response against 12 selected epitopes of PolyPEPI1018  
as measured by interferon (IFN)-gamma Enzyme-Linked ImmunoSpot (ELISPOT) assay for the following:  
o Number and proportion of subjects with 0, ≥1, ≥2, ≥3, ≥4, ≥5  
effector T cell responses, respectively, detected at the Baseline Visits (Week -3 to -1 and Week 0) and at 3, 6-, 9-, and 12-weeks  
after administration of the vaccine  
o For each subject, the number of effector T cell responses o For each subject, the time course of effector T cell response at  
Baseline Visits (Week -3 to -1 and Week 0) and at 3-, 6-, 9-, and 12-weeks after administration of the vaccine  
- Memory T cell response against 12 selected epitopes of PolyPEPI1018  
as measured by the Precursors with High Proliferation Capacity (PHPC) assay for the following:  
o Number and proportion of subjects with 0, ≥1, ≥2, ≥3, ≥4, ≥5  
memory T cell response, respectively, detected at the Baseline  
Visits (Week -3 to -1 and Week 0) and at 3-, 6-, 9-, and 12-weeks  
after administration of the vaccine  
o For each subject, the number of positive memory T cell responses  
o For each subject, the time course of memory T cell response at  
Baseline Visits (Week -3 to -1 and Week 0) and at 3-, 6-, 9-, and  
12-weeks after administration of the vaccine  
- Objective Response Rate (ORR) measured by CT scan at Screening  
Visit (Week -3 to -1), and at 6-, and 12-weeks after administration of each dose of the vaccine  
Exploratory Outcome Measures:  
- Correlations between PEPIs identified by the PEPI Test and effector and  
memory T cell responses measured by ELISPOT and PHPC  
immunogenicity assays  
- Change in relative counts of tumor infiltrating lymphocytes (TILs) in  
accessible tumor biopsies at Baseline (Week -3 to -1) and at Week 12 as  
measured by immunohistochemistry  
- Correlation between PEPIs identified by the PEPI Test and infiltrating  
lymphocyte in tumor biopsies measured by immunohistochemistry  
- Correlations between T cell responses measured by ELISPOT and  
PHPC and infiltrating lymphocyte in tumor biopsies measured by immunohistochemistry  
- Correlations between PEPIs identified by the PEPI Test and effector and  
ORR  
- Correlations between T cell responses measured by ELISPOT and  
PHPC and ORR  
Page 9 of 81  
| Sample Size Justification:   | Approximately 10 subjects will be enrolled in the study to receive three  vaccinations. The study is not statistically powered to assess the primary or  secondary outcomes measures. Assuming that the human leukocyte antigen (HLA) sets from the subjects  follow the distribution observed in the HLA sets from the in-house database,  at least 39 positive immune responses are expected from 10 subjects. The  distribution of the expected number of positive tests is displayed in  Figure S1, which is generated from 10,000 simulations. 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 least 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 displayed in  Figure S2, the x-axis shows the number of subjects with a given probability  Figures S2. Expected Distribution of Probabilities Having a Given  Number of Subjects with Positive Immune Response for Two or More   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
  
| Statistical Methods and   | Demographic and baseline characteristics will be summarized using                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
|---------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Analyses:                 | 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 change from Baseline in these parameters will  be summarized using descriptive statistics. |  
Page 10  
| 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.   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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clinical study protocol approval signature

Page  
| CLINICAL STUDY PROTOCOL                                                                                                                                     |                                                                                                                                    |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------|
| CLINICAL STUDY PROTOCOL APPROVAL SIGNATURE PAGE ..................................2                                                                         |                                                                                                                                    |
| INVESTIGATOR ACCEPTANCE PAGE .                                                                                                                              |                                                                                                                                    |
| S SYNOPSIS .                                                                                                                                                |                                                                                                                                    |
| TABLE OF CONTENTS                                                                                                                                           |                                                                                                                                    |
| LIST OF TABLES .                                                                                                                                            |                                                                                                                                    |
| LIST OF FIGURES .........................................................................................................................................15 |                                                                                                                                    |
| LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ....................................16                                                                       |                                                                                                                                    |
| 1                                                                                                                                                           | BACKGROUND INFORMATION .............................................................................................18             |
| 1.1                                                                                                                                                         | Colorectal Cancer Background                                                                                                       |
| 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                                                                                                                   |
| 2.1                                                                                                                                                         | Primary Objectives                                                                                                                 |
| 2.2                                                                                                                                                         | Secondary Objectives                                                                                                               |
| Exploratory Objectives ..................................................................................................28                                 |                                                                                                                                    |
| 2.3                                                                                                                                                         |                                                                                                                                    |
| 3                                                                                                                                                           | STUDY DESIGN                                                                                                                       |
| 3.1                                                                                                                                                         | Overall Study Design                                                                                                               |
| 3.2                                                                                                                                                         | OUTCOME MEASURES                                                                                                                   |
| Primary Outcome Measures                                                                                                                                    |                                                                                                                                    |
| 3.2.1                                                                                                                                                       |                                                                                                                                    |
| 3.2.2                                                                                                                                                       | Secondary Outcome Measures                                                                                                         |
| 3.2.3                                                                                                                                                       | Exploratory Outcome Measures ...............................................................32                                     |
| Estimated Study Duration ...........................................................................................32                                      |                                                                                                                                    |
| 3.3                                                                                                                                                         |                                                                                                                                    |
| 3.4                                                                                                                                                         | Randomization and Blinding ............................................................................................33          |
| 3.5                                                                                                                                                         | Study Stopping Rules                                                                                                               |
| 4                                                                                                                                                           | SUBJECT SELECTION .........................................................................................................34      |
| Inclusion Criteria .........................................................................................................34                              |                                                                                                                                    |
| 4.1                                                                                                                                                         |                                                                                                                                    |  
| Product: PolyPEPI1018 Colorectal Cancer Vaccine   | Page 12 of 81                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|---------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 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 PROCEDURES 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 Assessments 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                                                                                                                                                                                                               |  
Page 13 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 - |  
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
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8 adverse events and serious

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                            |  
OBERTO-101 Amendment 3  
| Product: PolyPEPI1018 Colorectal Cancer Vaccine                                                                                                  |                                                                                                                                      |
|--------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------|
| 8.2.1                                                                                                                                            | SEVERITY .                                                                                                                           |
| 8.2.2                                                                                                                                            | Causality ....................................................................................................................61     |
| 9                                                                                                                                                | STATISTICS .                                                                                                                         |
| 9.1                                                                                                                                              | Analysis Populations                                                                                                                 |
| 9.2                                                                                                                                              | Replacement of Subjects                                                                                                              |
| 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                                                                                                            |
| Primary Endpoint ..................................................................................................................63            |                                                                                                                                      |
| 9.4                                                                                                                                              |                                                                                                                                      |
| 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                   |
| Exploratory Analyses ............................................................................................66                              |                                                                                                                                      |
| 9.5.4                                                                                                                                            |                                                                                                                                      |
| 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                                                                                        |
| Confidentiality ...........................................................................................................................70    |                                                                                                                                      |
| 10.4                                                                                                                                             |                                                                                                                                      |
| 11                                                                                                                                               | ADMINISTRATIVE CONSIDERATION ...........................................................................71                           |
| 11.1                                                                                                                                             | Investigator's Responsibilities ............................................................................................71       |
| 11.2                                                                                                                                             | Study Training ...................................................................................................................71 |
| 1.3                                                                                                                                              | Study Monitoring ................................................................................................................71  |
| 11.4                                                                                                                                             | Safety Monitoring ................................................................................................................72 |
| 1.5                                                                                                                                              | A Audits                                                                                                                             |
| Documentation                                                                                                                                    |                                                                                                                                      |
| 11.6                                                                                                                                             | Case Report Forms and Study Records ...................................................72                                            |
| 11.6.1                                                                                                                                           |                                                                                                                                      |
| 11.6.2                                                                                                                                           | Direct Access to Source Data, Source Documents, and Study                                                                            |
| Records ......................................................................................................................................73 |                                                                                                                                      |
| 11.6.3                                                                                                                                           | SOURCE DATA                                                                                                                          |
| 11.6.4                                                                                                                                           | Document and Data Retention ...........................................................................74                            |
| Non-Compliance with the Protocol ...............................................................................74                               |                                                                                                                                      |
| 1.7                                                                                                                                              |                                                                                                                                      |  
Page 15  
| Product: PolyPEPI1018 Colorectal Cancer Vaccine   | Page 15 of 81              |
|---------------------------------------------------|----------------------------|
| 11.8                                              | Financing and Insurance 75 |
| 11.9                                              | Publication Policy 75      |
| 12                                                | REFERENCES 76              |  
APPENDIX A. SCHEDULE OF STUDY ASSESSMENTS ..................................78 APPENDIX B. **THE WORLD ALLERGY ORGANIZATION**
SUBCUTANEOUS IMMUNOTHERAPY SYSTEMIC REACTION GRADING SYSTEM .................................................................................................81
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list of tables

| Table 1.   | Synthetic Peptide Sequences 20     |
|------------|------------------------------------|
| Table 2.   | Local Reaction Assessment Scale 44 |
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list of figures

| Figure 1.   | Cancer Testis Antigens Targeted by PolyPEPI1018 21                                                                                  |
|-------------|-------------------------------------------------------------------------------------------------------------------------------------|
| Figure 2.   | Mechanism of Action of Precision PolyPEPI1018 CRC Vaccine 22                                                                        |
| Figure 3.   | Study Design Schema 30                                                                                                              |
| Figure 4.   | Distribution of the Expected Number of Positive Tests 67                                                                            |
| Figure 5.   | Expected Distribution of Probabilities Having a Given Number of Subjects  with Positive Immune Response for Two or More Antigens 68 |  
| LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS   |                                                |
|--------------------------------------------------|------------------------------------------------|
| Abbreviation                                     | Definition                                     |
| AE                                               | adverse event                                  |
| ALAT                                             | alanine amino transferase                      |
| ASAT                                             | aspartate amino transferase                    |
| CA 19-9                                          | cancer antigen                                 |
| CDx                                              | companion diagnostic                           |
| CEA                                              | carcinoembryonic antigen                       |
| CNS                                              | central nervous system                         |
| CPI                                              | checkpoint inhibitors                          |
| CRC                                              | colorectal cancer                              |
| CRF                                              | case report form                               |
| CTA                                              | cancer testis antigen                          |
| CTCAE                                            | Common Terminology Criteria for Adverse Events |
| CTL                                              | cytotoxic T lymphocyte                         |
| EC                                               | Ethics Committee                               |
| ECG                                              | electrocardiogram                              |
| ECOG                                             | Eastern Cooperative Oncology Group             |
| eCRF                                             | electronic case report form                    |
| EGFR                                             | epidermal growth factor receptor               |
| ELISPOT                                          | Enzyme-Linked ImmunoSpot                       |
| EU                                               | European Union                                 |
| 5FU                                              | 5-fluorouracil                                 |
| FDA                                              | Food and Drug Administration                   |
| GCP                                              | Good Clinical Practice                         |
| GMP                                              | Good Manufacturing Practice                    |
| hCG                                              | human chorionic gonadotropin                   |
| HLA                                              | human leukocyte antigen                        |
| ICF                                              | informed consent form                          |
| ICH                                              | International Council for Harmonisation        |
| IEC                                              | Independent Ethics Committees                  |
| IFN                                              | interferon                                     |
| IHC                                              | immunohistochemistry                           |
| IP                                               | investigational product                        |
| IRB                                              | Institutional Review Boards                    |
| mCRC                                             | metastatic colorectal cancer                   |
| MedDRA                                           | Medical Dictionary for Regulatory Activities   |  
| Product: PolyPEPI1018 Colorectal Cancer Vaccine   | Page 17 of 81                                                            |
|---------------------------------------------------|--------------------------------------------------------------------------|
| MEK                                               | MAPK/ERK kinase                                                          |
| MSI-H                                             | high microsatellite instability                                          |
| NCI-CTCAE                                         | National Cancer Institute-Common Terminology Criteria for Adverse Events |
| NMC                                               | non-medical complaint                                                    |
| ORR                                               | overall response rate                                                    |
| PBMC                                              | peripheral blood mononuclear cells                                       |
| PEPI                                              | personal epitope                                                         |
| PHPC                                              | Precursors with High Proliferation Capacity                              |
| PR                                                | partial response                                                         |
| RAS                                               | Rat Sarcoma Viral Oncogene Homolog                                       |
| RECIST                                            | Response Evaluation Criteria in Solid Tumors                             |
| RSI                                               | Reference Safety Information                                             |
| SAE                                               | serious adverse event                                                    |
| SAER                                              | SAE report                                                               |
| SAS                                               | Safety Analysis Set                                                      |
| SC                                                | subcutaneous(ly)                                                         |
| SIC                                               | subject identification code                                              |
| SoA                                               | Schedule of Study Assessments                                            |
| SOC                                               | system organ class                                                       |
| SUSAR                                             | suspected unexpected serious adverse reaction                            |
| TEAE                                              | treatment-emergent adverse event                                         |
| TIL                                               | tumor‐infiltrating lymphocyte                                            |
| ULN                                               | upper limit of normal                                                    |
| US                                                | United States                                                            |
| WT                                                | wildtype                                                                 |
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1 background information 1.1 colorectal

Colorectal cancer is a leading cause of cancer-related mortality worldwide. In the European Union (EU-28) and the World Health Organization Europe Region, the mean mortality rates per 100,000 population are respectively 15.2 and 15.7 for men and 9.0 and 9.7 for women (Ferlay 2015). The mean incidence rate of colorectal cancer in the EU-28 is 31.3 per 100,000 population (39.5 per 100,000 population for men and 24.4 per 100,000 population for women), with 345,000 new cases per year. Colorectal cancer is the third most common cancer and the third leading cause of cancer death in men and women in the United States (US). In 2010, the US spent $14 billion for the management of colorectal cancer (Mariotto 2011). While multidisciplinary care is employed in the treatment of localized colon cancer, systemic chemotherapy is the mainstay of treatment in metastatic colorectal cancer (mCRC). There are currently 10 Food and Drug Administration (FDA) approved drugs for the management of mCRC, including 5-fluorouracil (5FU), oxaliplatin, irinotecan, capecitabine, bevacizumab, cetuximab, panitumumab, ziv- aflibercept, regorafenib and ramucirumab. There are a variety of regimens with different combinations of these drugs, for use in first-, second-, and third-line settings of treatment. For example, 5FU, oxaliplatin (FOLFOX) and bevacizumab are commonly used as a first-line therapy (Saltz 2008). The Rat Sarcoma Viral Oncogene Homolog (RAS) is a family of genes including KRAS and NRAS that are often found to be mutated in colorectal cancer. When a tumor is RAS wildtype (WT), patients derive benefit from monoclonal antibodies, such as cetuximab or panitumumab, which target the epidermal growth factor receptor (EGFR). Thus, EGFR targeted therapy, in addition to chemotherapy, is a common second-line strategy for patients with RAS WT disease (Seymour 2013). The chemotherapy backbone used in second-line treatment is often 5FU and irinotecan (FOLFIRI), with regorafenib monotherapy commonly used as a third-line option (Grothey 2013). More recently it has been shown that FOLFOXIRI plus bevacizumab compared with FOLFIRI plus bevacizumab, improve the outcome in patients with mCRC (Loupakis 2014). Despite the available treatment options, response rates to mCRC remain low. Multiple therapies with novel mechanisms of actions are being developed to treat mCRC, including small molecule tyrosine kinase inhibitors, and MAPK/ERK kinase (MEK; also known as MAP2K) inhibitors. BRAF mutations, usually associated with poor prognosis, are found in ~10% of patients with colorectal cancer (CRC), and small molecule inhibitors of BRAF are being developed as combination treatments with other available therapies (Cassidy 2017). Immunotherapy is an attractive treatment option since it is more tolerable than conventional chemotherapy with potential long-lasting durable responses. Novel checkpoint inhibitors (CPIs) have demonstrated unprecedented clinical activity in a wide range of cancers. The observation of clinical activity in microsatellite instability-high (MSI-H) mCRC was the first indication of a potential for mCRC to respond to these agents, and has led to a breakthrough designation by the FDA for CPI use in this subset of patients (estimated as 5% to 10% of the patient population with mCRC). However, a proportion of MSI-H, and nearly all microsatellite stable disease, does not respond to single-agent checkpoint inhibition, and clinical trials are ongoing to increase responses to immunotherapy in mCRC through both better patient selection and novel combinations of immunotherapeutic agents (Bever 2017).
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1.2 polypepi1018 and companion diagnostic

PolyPEPI1018 CRC Vaccine (PolyPEPI1018) and companion diagnostic (CDx) product is a precision CRC vaccine that takes into account an individual patient's genetics and tumor heterogeneities using information technology during product design and responder selection processes. The subcutaneously (SC) injected vaccine product is a mixture of 6 synthetic peptides (Table 1) and the adjuvant Montanide™. The CDx is a pharmacogenetic test to be co-developed with PolyPEPI1018, which will select patients who most likely respond to vaccine treatment. The CDx uses the complete human leukocyte antigen (HLA) genotype of the patient for the identification of personal epitopes (PEPIs) that induce T Cell responses. To identify PEPIs the sponsor developed the novel PEPI Test. As described in Section 6.6.1, the PEPI Test selects, from HLA restricted epitopes, certain PEPIs that are capable of activating T cells in an individual. PEPIs are genetic biomarkers specific to the HLA genotype of an individual patient. The PolyPEPI10108 CDx will use the PEPI Test to identify the patient's PEPIs from PolyPEPI1018. Data from a retrospective correlation study performed by the sponsor suggest that patients with PEPIs derived from multiple CRC antigens are likely to benefit from PolyPEPI1018 vaccination.
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table 1. synthetic peptide sequences

| ID     | Sequence                       |
|--------|--------------------------------|
| CRC_P1 | PSTTMETQFPVSEGKSRYRAQRFWSWVGQA |
| CRC_P2 | VRTYWIIIELKHKARTAKKVRRAIEQLAAM |
| CRC_P3 | YVDEKAPEFSMQGLKDEKVAELVRFLLRKY |
| CRC_P6 | PKSMTMMPALFKENRSGAVMSERVSGLAGS |
| CRC_P7 | KFMNPYNAVLTKKFQKVNFFFERIMKYERL |
| CRC_P8 | AQKMSSLLPTMWLGAFKKTMSTFHNLVSLN |  
The 6 peptides in PolyPEPI1018 contain 12 novel and distinct selected epitopes which are derived from 7 conserved cancer testis antigens (CTAs) that act in combination to activate T cells against CRC antigens in a high proportion of patients. The 7 most frequently expressed CTAs in CRC were selected as targets of PolyPEPI1018, and statistical analysis demonstrates a 95% probability that 3 of these 7 CTAs are expressed in any CRC cell (Figure 1). Given the high probability of targeting CRC expressed CTAs by PolyPEPI1018, tumor biopsy and CTA expression analysis, a risky, inaccurate and costly procedure1, is not required prior to vaccination with PolyPEPI1018.
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figure 1. cancer testis antigens

Abbreviations: CRC = colorectal cancer; CTA = cancer testis antigen Left: Expression rate in CRC obtained from 2,391 CTA expression analyses in tumor specimens.  
Right: Probability of expression in CRC tumor shows that 3 out of 7 CTAs are expressed in each tumor with
>95% probability.
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1.2.1 mechanism of action

Cancer vaccines represent the next generation immunotherapies (Chen 2013). They induce specific immune responses against tumor antigens, thereby boosting the immune system to attack cells expressing the targeted antigens. Tumor antigens that are currently being targeted by available immunotherapies include MUC1 and NY-ESO-1. Though current cancer vaccines have an excellent safety profile, the response rate is low due to poor antigen selection and the absence of a biomarker for identification of likely responders. PolyPEPI1018 induces PEPI-specific immune responses against multiple CTAs that are expressed in CRC and are not typically expressed beyond embryonic development in healthy cells. One PEPI derived from one tumor antigen can activate one cytotoxic T lymphocyte (CTL) to proliferate (clonal expansion) and kill tumor cells that present the same PEPI. If the patient has a homogenous tumor that consists of a single type of cell which presents the same PEPI, treatment with a single peptide vaccine could be successful. However, most tumors, especially at the metastatic stage, represent a fast-evolving group of heterogeneous tumor cells which differ in antigen expression and PEPI presentation. Therefore, boosting several CTL clones (i.e., polyvalent immune responses) using a combination of vaccines increases the probability of tumor cell recognition and tumor killing (Figure 2). Figure 2. Mechanism of Action of Precision PolyPEPI1018 CRC Vaccine  
  
Abbreviations: CTL = cytotoxic T lymphocyte; HLA = human leukocyte antigen; PEPI = personal epitope A: One peptide in the vaccine is processed to PEPI in the body of the patient by antigen presenting cells (APC).  
The patient's HLA molecules on the APC bind and present the PEPI to activate a CTL. This CTL proliferates and differentiates to a PEPI-specific CTL.  
B: A PEPI-specific CTL is able to recognize and destroy the tumor cells presenting the same PEPI. C: PolyPEPI1018 is based on the combination of several vaccines capable to induce multiple different PEPI-specific CTLs.  
D: These CTLs may recognize and destroy heterologous tumor cells in the patient presenting the same PEPIs.
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1.2.2 preclinical and clinical background

PolyPEPI1018 will be evaluated in patients with mCRC. Since this is a first-in-human study, clinical safety and efficacy data from other peptide vaccines are summarized in the sections below.
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1.2.2.1 safety of peptide vaccines

Overall there is a well-established safety profile for peptide vaccines and the adjuvant Montanide™. To date, there are 22 published clinical trials, in 813 patients, on peptide vaccines formulated with the adjuvant Montanide™ (details are provided in the Investigator Brochure). The most common side effect of peptide vaccines is inflammation at the injection site, including redness, pain, swelling, warming of the skin, itchiness, and rash. Occasionally, but rarely, unacceptable local skin reactions developed after 10 or more vaccinations, causing treatment to be terminated. After receiving a peptide vaccine, patients occasionally experienced flu-like symptoms, including fever, chills, weakness, dizziness, nausea or vomiting, muscle ache, fatigue, headache, and occasional breathing difficulties. Blood pressure was also affected in some subjects who received a peptide vaccine. Many of these side effects, which last for a short time, are signs/symptoms of inflammation (similar to those occurring after an acute viral infection), likely indicating that the patient was mounting an immune response. Peptide vaccines, like any other medication affecting the immune system, can cause adverse effects that may prove life threatening. For example, severe hypersensitivity (allergic) reactions to specific vaccine ingredients have occurred following vaccination; however, such severe reactions are very rare (Yoshida 2011). Furthermore, the design of PolyPEPI1018 excludes peptides containing self-epitopes, thus reducing to a minimum the risk of autoimmune and/or hypersensitivity reactions. In the 813 subjects from the 22 published clinical trials on peptide vaccines formulated with Montanide™ the most serious adverse events (SAEs) were caused by cancer progression. Vaccine-related Grade 3 events were very rare (6 events, 0.7%), and consisted of skin reactions or cellulitis at the injection site, edemas of the head and neck regions, colitis, rectal bleeding and bladder-vaginal fistulae.
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1.2.2.2 efficacy of peptide vaccines

PolyPEPI1018 belongs to the class of peptide vaccines. Previous clinical studies have demonstrated that peptide vaccines induce CTL responses. However, the main limitation of these peptide vaccines is the absence of the induction of CTL responses against multiple antigens expressed in tumor cells. The sponsor hypothesized that induction of antigen specific immune response against multiple antigens that are expressed by the tumor cells will substantially improve the immune response rate and efficacy of the cancer vaccine. Consistent with this expectation, preliminary clinical results with IMA901 peptide vaccines demonstrated that polyvalent tumor specific CTL responses led to clinical benefit in renal cancer patients (Walter 2012); two independent studies with personalized cancer vaccines confirmed these findings in melanoma patients (Ott 2017; Sahin 2017). A retrospective clinical study conducted by the sponsor demonstrated significant correlation between objective response rate (ORR) of cancer vaccines containing multiple antigens and PEPIs on at least 2 vaccine antigens (see the Investigator Brochure). These data suggest that that patients with PEPIs derived from multiple CRC antigens are likely to benefit from PolyPEPI1018 vaccinations.
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1.3 rationale for study design

Treos Bio ZRT (sponsor) has developed a vaccine, PolyPEPI1018, which has the potential to elicit an immune response in most individuals with mCRC. Such an immune response is expected to control, reduce, or eliminate cancer in those patients. The sponsor elected to move from a single vaccination (as originally planned) to multiple (N=3) vaccinations during this first-in-human study to establish the rate, intensity, and evolution of immune responses to PolyPEPI1018 as well as initial efficacy. Since a sufficient time (typically 3 months/12 weeks) is required after vaccination to observe the time course of both effector and memory T cell responses, the maintenance phase following first-line therapy of mCRC (a 4 to 6 month period when the tumor, therapy, and patient's condition are expected to remain stable [Heinemann 2014; Cremolini 2015; Venook 2017]) is the preferred period for treatment with PolyPEPI1018 since it offers optimal conditions for monitoring the time course of immune response after at least the initial vaccination. If a progression occurs during the course of the trial the subject will still be immunized at the expected times (Weeks 13 and 26), even when a second line therapy is needed and administered. Understanding the evolution of T cell response after vaccination will provide valuable information to design the vaccine administration schedule in future clinical studies. Moreover, a clean readout of immune responses after single vaccination with adequate follow-up will allow a better assessment of whether the PEPI Test can accurately predict immune responses. Clarifying such correlation will provide a predictive tool that can be used as a CDx in future trials. Therefore, multiple vaccinations 13 week apart with a 12-week follow-up period after third and final vaccination will provide information to design subsequent Phase 2/3 trials. Only 1 line of prior mCRC chemotherapy treatment is allowed for subjects in this study since response to second- and third-line therapy is often associated with worse and shorter response compared with first-line therapy. Therefore, allowing more than 1 line of prior chemotherapy would not fulfil the expectation of stable conditions during the 12-week follow-up period after first vaccination. Male or female subjects must be between 18 years and 75 years of age at time of Screening since a robust immune system is needed to elicit an appropriate immune response to vaccination with PolyPEPI1018. Also for this reason, subjects must have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. The sponsor intends to explore whether multiple vaccinations are able to elicit an immune response that is sufficiently robust to generate tumor infiltrating lymphocytes (Klintrup 2005), and whether this correlates with PEPIs. The sponsor also intends to explore the initial efficacy of PolyPEPI1018 multiple vaccinations. Consequently, subjects must have histologically confirmed metastatic adenocarcinoma originating from the colon or the rectum, with the presence of at least 1 measurable reference lesion according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 criteria. The progression of CRC during the 12 weeks following each vaccination will be monitored by CT scan every 6 weeks not only to measure efficacy but also to establish whether subject need to start a second line induction therapy. Subjects in the study will receive maintenance therapy with a fluoropyrimidine (5fluorouracil or capecitabine) plus the same biologic agent (bevacizumab, cetuximab or panitumumab) used during induction. It is typically assumed that chemotherapy is immune suppressive; however, recent advances have shown that in several instances chemotherapy might exert positive effects on immune responses by inducing mechanisms that potentiate immunity (Emens 2015). It is also anticipated that treatment with a biologic drug during maintenance therapy will not interfere with vaccination. Studies have shown that EGFR inhibitors do not interfere with immunotherapies and bevacizumab potentiates immune responses (Terme 2013; Mansfield 2013). Therefore, subjects will be immunized even when a second line therapy is administered. Finally, maintenance therapy is allowed with 1 biological therapy regimen with or without a systemic chemotherapy regimen scheduled to initiate prior to vaccination and to continue throughout the 12-week follow-up period. Thus, patients may receive standard-of-care treatment while participating in this study, including introducing a second line therapy, if needed.
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1.4 benefits and risks

It is expected that PolyPEPI1018 will induce PEPI-specific immune responses against multiple CTAs expressed in CRC cells. Previous clinical studies have demonstrated that peptide vaccines similar to PolyPEPI1018 induce CTL responses. Recently, 2 independent studies have shown that personalized vaccination of melanoma patients, with selected mutant proteins identified as being the most likely to generate an immune response, resulted in atypically long relapse-free durations after vaccination (Ott 2017; Sahin 2017). Limitations of most vaccines include a lengthy period needed to identify the targets, formulate and manufacture vaccines. The present study would provide information for vaccine improvement. As described in Section 1.2, PolyPEPI1018 consists of the 7 most frequently expressed CTAs in CRC, and there is a 95% probability the 3 of these 7 CTAs are expressed in any CRC cell. It is expected that PolyPEPI1018 will induce cytotoxic T cell responses against CTAs expressed in CRC cells, thereby matching the vaccines to patient's tumor antigens and whole HLA genotype. Only a saliva sample, not the traditionally required tumor biopsy, will be required for performing the PEPI Test for the identification of likely responders, thus simplifying and accelerating the selection procedure. In order to examine whether the PEPI Test can predict immune responses and identify potential responders (thus providing the basis and the rationale for its use as CDx in future studies) it is essential that the present study allows 12 weeks of follow-up after each vaccination. This will also provide useful information to inform us on scheduling (how often vaccination should be repeated) during future multiple-vaccination studies. As described in Section 1.2.2.1, based on the safety profile of similar vaccines, PolyPEPI1018 is expected to be safe and well-tolerated. Side effects are typically mild (Section 1.2.2.1.). Local erythema and edema in the site of vaccination are expected, as well as a flu-like syndrome with minor fever and fatigue. Vaccines, like any other medication that affects the immune system, can cause adverse effects that may prove life threatening. For example, severe hypersensitivity (allergic) reactions to specific vaccine ingredients have occurred following vaccination; however, such severe reactions are very rare (Yoshida 2011). Furthermore, the design of PolyPEPI1018 excludes peptides containing self-epitopes, thus reducing the risk of autoimmune and/or hypersensitivity reactions to a minimum.
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2 study objectives 2.1 primary

The primary objective of this study is:  
- To evaluate the safety and tolerability of a multiple doses of PolyPEPI1018 as an addon to maintenance therapy in subjects with metastatic colorectal cancer (mCRC)
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2.2 secondary objectives

The secondary objectives of this study are:  
- To identify PEPIs (Personal EPItopes capable of inducing T cell responses in an individual) from PolyPEPI1018 in each study subject
- To evaluate the immunogenicity of PolyPEPI1018 by measuring both effector and memory T cell responses
- To evaluate initial efficacy of PolyPEPI1018 by evaluating Objective Response Rate
(ORR)
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2.3 exploratory objectives

The exploratory objectives of this study are:
- To explore the correlation between PEPIs and T cell responses  
- To explore the correlation between PEPIs and tumor infiltrating lymphocytes
- To explore the correlation between T cell responses and tumor infiltrating lymphocytes
- To explore the correlation between PEPIs and ORR
- To explore the correlation between T cell responses and ORR
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3 study design 3.1 overall

This is a Phase 1, open-label, non-randomized, multicenter study to evaluate the safety, tolerability, immunogenicity and efficacy of multiple subcutaneous (SC) injections of PolyPEPI1018 as an add-on immunotherapy to the standard-of-care maintenance therapy in approximately 10 subjects with mCRC. This is an amended Study Design only in the USA: 5 subjects in Italy are under evaluation after only a single SC injection of the same vaccine, as originally planned. The study is composed of a 3-week Screening Period, the administration of multiple doses vaccine (Day 1, Week 0; Day 92, Week 13; Day 183, week 26), a 13-week Follow-up Period between the first and second vaccination and a 12-week Follow-up Period after the last vaccination. The study will be conducted on an outpatient basis. The Screening Period will occur between 21 days and 1 day prior to administration of PolyPEPI1018 administration. Screening should be performed in parallel with the subject's completion of the standard-of-care first-line treatment and initiation of the standard-of-care maintenance treatment. During this study period, results from a CT scan, which should be performed at the completion of the first-line treatment regimen, must be obtained. The results from the CT scan must confirm that the subject has responded to first-line chemotherapy with documentation of cancer regression or lack of progression, according to the RECIST version 1.1 criteria. Once informed consent has been obtained, the subject will undergo a biopsy procedure in one of the accessible tumors (primary or metastasis) to check for tumor infiltrating lymphocytes (TILs). The first dose of PolyPEPI1018 will be administered on Day 1 of Week 0 and must be (1) after the subject initiates the maintenance regimen, and (2) within 3 weeks after the eligibility CT scan was performed. Vaccination will be performed at 4 anatomic sites (preferably draining to axillary or inguinal lymph nodes) and administered by SC injection. After the last injection, the subject must remain at the site for at least 1 hour to be monitored for any local and/or systemic allergic reactions. A buccal swab will be collected to determine HLA types. A subject diary will be distributed to subjects to collect local signs related to the injection sites, physical fatigue, and body temperature. Subjects will be monitored every 3 weeks for 12 weeks after first administration of PolyPEPI1018 (i.e. Weeks 3, 6, 9 and 12), then vaccinated again at week 13 and monitored every 3 weeks for 12 weeks after second administration of PolyPEPI1018 (i.e. Weeks 16, 19, 22 and 25), then vaccinated again at week 26 and monitored every 3 weeks for 12 weeks after third administration of PolyPEPI1018 (i.e. Weeks 29, 32, 35 and 38). The timing of the study assessments and procedures are summarized in the Schedule of Study Assessments (SoA) in Appendix A.
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figure 3. study design schema

Five subjects in Italy will follow the original Study Design Ten Subjects in the USA will follow the amended Study Design; those who have already  
 signed an Informed Consent Form for the original Study Design will be asked to sign a new Informed Consent Form at the end of the first 12-week follow-up period.
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3.2 outcome measures

3.2.1 **Primary Outcome Measures**  
- The incidence and severity of all adverse events (AEs), related AEs, all SAEs, related SAEs, and temporally-associated AEs according to the National Cancer InstituteCommon Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0 (v4.0)
- Change from baseline in clinical laboratory safety parameters and vital signs  
- Number and proportion of subjects with any clinically significant change in vital signs (i.e., blood pressure, pulse rate, respiratory rate, body temperature) during the vaccine administration or within 60 minutes following administration
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3.2.2 secondary outcome measures

- PEPIs as identified by the PEPI Test
- Effector T cell response against 12 selected epitopes of PolyPEPI1018 as measured by interferon (IFN)-gamma Enzyme-Linked ImmunoSpot (ELISPOT) assay for the following:
o Number and proportion of subjects with 0, ≥1, ≥2, ≥3, ≥4, ≥5 effector T cell responses, respectively, detected at the Baseline Visits (Week -3 to -1 and Week 0) and at 3, 6-, 9-, and 12-weeks after administration of the vaccine o For each subject, the number of effector T cell responses o For each subject, the time course of effector T cell response at Baseline Visits
(Week -3 to -1 and Week 0) and at 3-, 6-, 9-, and 12-weeks after administration of the vaccine
- Memory T cell response against 12 selected epitopes of PolyPEPI1018 as measured by the Precursors with High Proliferation Capacity (PHPC) assay for the following:
o Number and proportion of subjects with 0, ≥1, ≥2, ≥3, ≥4, ≥5 memory T cell response, respectively, detected at the Baseline Visits (Week -3 to -1 and Week 0) and at 3-, 6-, 9-, and 12-weeks after administration of the vaccine o For each subject, the number of positive memory T cell responses o For each subject, the time course of memory T cell response at Baseline Visits
(Week -3 to -1 and Week 0) and at 3-, 6-, 9-, and 12-weeks after administration of the vaccine
- Objective Response Rate (ORR) measured by CT scan at Screening Visit (Week -3 to
-1), and at 6-, and 12-weeks after administration of each dose of the vaccine
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3.2.3 exploratory outcome measures

- Correlations between PEPIs identified by the PEPI Test and effector and memory T cell responses measured by ELISPOT and PHPC immunogenicity assays
- Change in relative counts of TILs in accessible tumor biopsies at Baseline (Week -3 to -1) and at Week 12 as measured by immunohistochemistry
- Correlation between PEPIs identified by the PEPI Test and infiltrating lymphocyte in tumor biopsies measured by immunohistochemistry
- Correlations between T cell responses measured by ELISPOT and PHPC and infiltrating lymphocyte in tumor biopsies measured by immunohistochemistry
- Correlations between PEPIs identified by the PEPI Test and effector and ORR
- Correlations between T cell responses measured by ELISPOT and PHPC and ORR
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3.3 estimated study duration

Four out of ten subjects have already been accrued under the original single-dose Study Design, the first one being accrued in May 2017. These subjects will be asked to sign a new informed consent for multiple dosing and it is anticipated that they will be willing to agree. It is also anticipated that subject accrual rate for the remaining six subjects will be one subject every 20 days. Based on this rate it will take approximately 4 months to accrue a total of 10 subjects. Since subjects are followed for 38 weeks after the PolyPEPI1018 initial vaccination, the total duration of the study is approximately 14 months from the date of amendment, that is approximately 17 months from the initiation of the OBERTO study.
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3.5 study stopping rules

The sponsor has the right to terminate the study at any time. The study will be stopped if any of the following occur:  
- New information leading to an unfavorable risk-benefit judgement of PolyPEPI1018, including the following (applicable after every vaccination and every five patients):
o 3 or more DLTs within 21 days of peptide vaccine administration o 1 or more deaths (other than death related to progressive disease) that occurs within 30 days of peptide vaccine administration o Other unfavorable safety findings
- Sponsor's decision that continuation of the study is unjustifiable for medical or ethical reasons
- Poor enrollment of subjects making completion of the study within an acceptable time frame unlikely
- Discontinuation of the development of PolyPEPI1018
- Specific request of a health authority
In accordance with applicable regulations, health authorities and Independent Ethics Committees (IECs)/Institutional Review Boards (IRBs) will be informed by the sponsor if the study is discontinued. If a study is discontinued, the investigator will inform the subject's primary health care provider, as applicable, to ensure the subject receives the appropriate follow-up and care.
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4 subject selection

A total of 10 subjects with mCRC who have achieved partial response (PR) or stable disease during first-line treatment with chemotherapy plus a biologic drug will be enrolled.
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4.1 inclusion criteria

Subjects who meet ALL of the following criteria are eligible for this study:  
1. Male or female subjects, 18-75 years of age at time of Screening who provide written informed consent prior to initiation of any study procedure 2. Histologically confirmed metastatic adenocarcinoma originating from the colon or the rectum 3. Presence of at least 1 measurable reference lesion according to the RECIST
version 1.1 criteria 4. Experienced PR or stable disease during first-line treatment with a systemic chemotherapy regimen and 1 biological therapy regimen 5. Maintenance therapy with a fluoropyrimidine (5-fluorouracil or capecitabine) plus the same biologic agent (bevacizumab, cetuximab or panitumumab) used during induction, scheduled to initiate prior to the first day of treatment with the study drug 6. No more than 1 line of chemotherapy regimen for mCRC (adjuvant therapy for non-metastasized disease is allowed if terminated more than 6 months before Screening and without recurrence within 6 months after the end of adjuvant treatment)
7. Last CT scan at 3 weeks or less before the first day of treatment 8. ECOG performance status of 0 or 1 9. Women of childbearing potential must agree to appropriately use an effective form of contraception (failure rate of <1% per year) for 3 months from the day of the treatment. An effective form of contraception is defined as using hormonal contraceptives or an intrauterine device combined with at least 1 of the following forms of contraception: a diaphragm, cervical cap or condom 10. Men must agree to use an effective form of contraception (as defined above), and not donate sperm for 3 months from the day of the treatment 11. White blood cell count ≥3.0 × 109/L with neutrophils ≥1.5 × 109/L 12. Platelets ≥100 × 109/L, hemoglobin ≥5.6 mmol/L (corresponding to 9 g/dL)
13. Serum bilirubin ≤1.5 × upper limit of normal (ULN) set by the site  
14. Alanine amino transferase (ALAT) and aspartate amino transferase (ASAT)
≤2.5 × ULN in the absence of liver metastases. ALAT and ASAT ≤5 × ULN set by the site in the presence of liver metastases 15. Serum creatinine ≤1.5 × ULN set by the site and creatinine clearance >30 mL/min using Cockroft formula 16. Relevant toxicities of prior therapies must have resolved, except for oxaliplatin-related neuropathy or alopecia 17. Anticipated life expectancy ≥6 months 18. Subject is willing and able to comply with the requirements of the protocol
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4.2 exclusion criteria

Subjects who meet ANY of the following criteria are not eligible for this study:  
1. Received chronic systemic immune therapy or immunosuppressant medication other than steroids within the last 6 weeks prior to start of study treatment 2. Received continuous systemic steroid treatment within the last 2 weeks prior to start of study treatment 3. Colorectal cancer with documented MSI-H 4. Colorectal cancer with documented BRAF mutations 5. Pre-existing systemic autoimmune or antibody-mediated diseases or immune deficiency diseases 6. Central nervous system (CNS) metastases 7. Active or uncontrolled severe infections or undiagnosed febrile condition >38ºC 8. Acute or subacute intestinal obstruction or history of chronic intestinal inflammatory diseases 9. Symptomatic peritoneal carcinomatosis 10. Peritonitis 11. Serious, non-healing wounds, ulcers or bone fractures 12. Nephrotic syndrome 13. Arterial thromboembolisms or severe hemorrhages within 6 months before study enrolment (except bleeding tumor before tumor resection surgery)
14. Hemorrhagic diathesis or thrombotic tendency 15. Major surgery or radiotherapy within 12 weeks prior to the study treatment or anticipation of needing such procedure during the study period 16. Uncontrolled pleural effusion, pericardial effusion or ascites requiring repeated drainage more than once every 28 days 17. Participants with active malignancy (other than CRC) or a prior malignancy within the past 12 months 18. Participant with myocardial infarction within 6 months prior to enrollment or New York Heart Association Class III or IV heart failure, uncontrolled angina, severe uncontrolled ventricular arrhythmias or electrocardiographic evidence of acute ischemia or active conduction system abnormalities. Prior to the first dose of study treatment, any electrocardiogram (ECG) abnormality at screening must be documented by the investigator as not medically relevant 19. Administration of a live, attenuated vaccine within 4 weeks before randomization or anticipation of a live attenuated vaccine will be required during the study 20. Participant has participated in another clinical study involving an investigational product (IP) or investigational device within 30 days prior to enrollment or is scheduled to participate in another clinical study involving an IP or investigational device during the course of this study 21. Known hypersensitivity to any component of the investigational drug 22. If female, participant is pregnant (exclusion confirmed with beta-human chorionic gonadotropin [hCG] test) or lactating at the time of enrollment, or has plans to become pregnant or start breastfeeding during the study 23. Pre-existing alcohol or drug abuse 24. Medical or mental impairments which make it impossible to obtain the patient's consent or to conduct the study 25. A significant concomitant medical condition which the clinical investigator believes precludes the patient from enrolling in the study 26. Absent or limited legal competence
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4.3 subject withdrawal and discontinuation

Any subject may voluntarily withdraw consent (i.e., reduce the degree of participation in the study) for aspects of continued participation and data collection. The reason for withdrawal will be recorded on the End of Study Case Report Form (CRF). Assessments to be performed at the Last Visit are described in Section 7.4. Discontinuation (i.e., complete withdrawal from study participation) may be due to dropout (i.e., active discontinuation by subject) or loss to follow-up (i.e., discontinuation by subject without notice or action). Additionally, the investigator and sponsor have the discretion to discontinue any subject from the study if, in their judgment, continued participation would pose an unacceptable risk for the subject. During the initial study period, that is during the 12 weeks following the first vaccination, if a patient experiences disease progression and needs to start a second-line therapy, the patient will be withdrawn from the study. This is required to obtain a complete (12 weeks) measurement of the immunological response curve after the initial vaccination, that is one of the original study objectives. In contrast, if a patient experiences disease progression and needs to start a second-line therapy after the second vaccination, the patient will remain in the study, receive the third vaccination as scheduled and complete follow-up. This will allow to gain a better sense of initial efficacy of the PolyPEPI1018 vaccine, that is another study objective. If a subject must be withdrawn from the study, the responsible designee medical monitor will be informed immediately, and this person will then notify the sponsor's medically responsible party. All subjects who prematurely discontinue from the study, regardless of cause, should be seen as soon as possible and undergo the assessments specified for the Last Visit (see Section 7.4). If there is a medical reason for withdrawal, the subject will remain under the supervision of the investigator until satisfactory health has returned or health has stabilized. The investigator will inform the subject's primary health care provider (as applicable) as agreed by the subject, to ensure the subject receives the appropriate follow-up and care. If the subject is prematurely discontinuing due to withdrawal of consent, the investigator should clarify the level of consent withdrawal with the subject (i.e., consent withdrawal with regard to permission to utilize samples already collected but not analyzed and willingness to return for safety follow-up assessments). The level of consent withdrawal should be documented this in the electronic case report form (eCRF) For subjects who are lost to follow-up (i.e., those subjects whose status is unclear because they fail to appear for study visits without stating an intention to withdraw), the investigator should show "due diligence" by documenting in the source documents all steps taken to contact the subject (e.g., dates of telephone calls and registered letters). Subjects who discontinue or are withdrawn from the study, including withdrawal after receipt of the study's IP on or after Day 1 of Week 0, will be replaced, except for subjects who experience a DLT: a subject who experiences a DLT will be withdrawn from the study and will not be replaced.
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4.3.1 permanent treatment discontinuation

Subjects will be permanently withdrawn from treatment for the following reasons:  
- Vaccine-related toxicity (Section 5.5)
- Requirement of prohibited concomitant medications (Section 5.4.2)
- Request by subject to terminate study - Completion of the study treatment as defined by the protocol
- Clinical reasons believed life threatening by the physician, even if not addressed in Section 5.5
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4.3.2 premature treatment discontinuation

Subjects will be prematurely discontinued from treatment for the following reasons:
- Failure by the subject to attend two consecutive clinic visits  
- Request by the subject to withdraw
- Pregnancy
- Development of an exclusionary condition as outlined in Section 4.2 - Request of the primary care provider if s/he thinks the study is no longer in the best interest of the subject
- Subject judged by the investigator to be at significant risk of failing to comply with the provisions of the protocol as to cause harm to self or seriously interfere with the validity of the study results
- Subject reaches a defined study endpoint, as outlined in Section 3.2, if applicable - At the discretion of the health authority, IRB/IEC, investigator, or pharmaceutical supporter
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5.1.1.1 vaccine substance

The vaccine is composed of 6 synthetic peptides and is manufactured according to Good Manufacturing Practices (GMP) using solid phase peptide synthesis. The vaccine substance should be stored in vials as a peptide solution (0.2 mg/mL in DMSO/water) at -20ºC.
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5.1.1.2 adjuvant

The adjuvant Montanide™ (ISA 51 VG) is a mixture of highly purified mineral oil and mannide monooleate (derived from vegetable grade olive oil). It is manufactured according to GMP and should be stored in vials as an emulsion at room temperature.
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5.1.2 polypepi1018 product preparation and

PolyPEPI1018 final vaccine product will be prepared at the clinical site prior to injection by the physician or the nurse according to standard operating procedure and should be administered as soon as possible following preparation. The peptide solutions (see Section 5.1.1.1) should be thawed and incubated at room temperature with the adjuvant Montanide™. A 4-mL volume of the final PolyPEPI1018 product will be prepared by mixing (emulsifying) each of the peptide solutions with an equal volume of the adjuvant Montanide™. Each dose of the final PolyPEPI1018 product will contain 0.8 mg peptides per 1 mL (4 peptides) solution + 0.4 mg peptides per 1 mL (2 peptides) solution plus 2 mL adjuvant. Prior to the PolyPEPI1018 administration, the subject's body temperature should be checked and the vaccine should only be administered if the subject's body temperature is <38ºC. Each subject will receive a total of four 1 mL SC injections of the final PolyPEPI1018 product (i.e., one 1 mL SC injection per predetermined anatomic site, (preferably draining to axillary or inguinal lymph nodes). A pause of a few minutes should be observed after the first injection to check for side effects. The remaining injections can be subsequently administered without pause. Additional details are provided in the Study Procedures Manual.
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5.1.3 packaging and labeling

PolyPEPI1018 peptide solutions will be supplied in pre-labeled boxes by the sponsor and the labels will be compliant with country specific regulatory requirements. Each box will contain vials of PolyPEPI1018-Mix1 and vials of PolyPEPI1018-Mix2 that needs to be stored in the freezer (-20oC). Each box will contain a label firmly attached to the box, with the company name, lot number, storage temperature, expiry date and a statement that the box contains vials of PolyPEPI1018-Mix1 and PolyPEPI1018-Mix2, respectively. Vials themselves will have label containing the vaccine name and lot number, content of the vial, strength of the solution, route of administration and storage temperature. One vial of each box will be used per subject for the preparation of the vaccine product at the clinical site. Adjuvant Montanide™ will be shipped by Seppic in pre-labeled boxes that need to be stored at room temperature (15ºC to 30ºC). Each box will contain 3 mL vials labeled as Montanide™ ISA 51 VG sterile, lot number, composition, manufacturer and expiration date. One vial will be used per peptide mixture, 2 vials per subject.
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5.1.4 supply distribution and pharmacy

The following study supplies are needed and will be provided as described below:
- PolyPEPI1018 vials will be distributed by the sponsor as described in Section 5.1.3.  
- Montanide™ adjuvant will be distributed by the supplier, Seppic SA, as described in Section 5.1.3.  
- Single use plastic equipment for PolyPEPI1018 vaccine emulsification procedure will be supplied by the Sponsor. Syringes, I-connector and vial adapters will be packaged together without harming the individual sterile packing of the devices. Each set will contain enough supplies for one emulsification procedure.  
Each package contains:
o 2 pieces of Disposable latex, silicone and rubber free syringe (3 ml B.Braun, Injekt® Solo, VWR cat nr.: 720-2522).  
o 1 piece of I-connector (Double Female Luer Lock Adapter, Promepla, Ref:
ODG0015ST).  
o 2 pieces of Vial adaptors (West Pharma, 13 mm, Luer Lock, sterile).
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5.1.5 storage 5.1.5.1 polypepi1018

Intact vials containing PolyPEPI1018 should be stored between -25ºC and -15ºC. Thawed vials may be stored for up to 3 hours at room temperature. Re-freezing is not allowed. Records must be available showing periodic temperature checks of storage freezer material. Vaccine emulsions consisting of peptide admixed with Montanide™ are stable for 4 hours at 4ºC, but should be administered as soon as possible following preparation.
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5.3 investigational product accountability

The investigator will ensure that the IP (i.e., PolyPEPI1018 and Montanide™) is stored as specified in Section 5.1.5 and that the storage area is secured, with access limited to authorized study personnel. The investigator will maintain records that the IP was received, including the date received, drug identity code, date of manufacture or expiration date, amount received and disposition. The IP must be dispensed only at the study site or other suitable location. Records will be maintained that includes the subject identification code (SIC), dispensation date, and amount dispensed. All remaining partially used and/or unused IP(s) will be returned to the sponsor or sponsor's representative after study completion/termination, or destroyed with the permission of the sponsor in accordance with applicable laws and study site procedures. If IP(s) is to be destroyed, the investigator will provide documentation in accordance with sponsor's specifications.
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5.4 concomitant medications

A concomitant medication is defined as any prescription or over-the-counter preparation, including vitamins and supplements. All concomitant medications from the initiation of the first-line CRC therapy until 85 days after the subject's vaccination must be recorded on the eCRF.
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5.4.1 required medications

Subjects must be on maintenance therapy with a fluoropyrimidine (5-fluorouracil or capecitabine) plus the same biologic agent (bevacizumab, cetuximab or panitumumab) used during induction, prior to Day 1 (Week 0, Visit 2). Modifications may be allowed for toxicity/tolerability reasons, but must be approved by the medical monitor to determine whether the subject can remain on study treatment/on study.
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5.4.2 prohibited medications

The concomitant use of the following immunomodulator therapies, which have potential putative effects on immunologic and/or virologic indices, are prohibited while on study:
- Systemic (intravenous and oral) corticosteroids
- Thalidomide
- Cyclosporine
- Interferons
- Interleukins
- IgG-containing products
- Cimetidine (Tagamet) - Acetylcysteine (NAC)
- Sargramostim (GM-CSF)
- Dinitrochlorobenzene (DNCB) - Thymosin alpha 1 (thymosin alpha)
- Thymopentin
- Inosiplex (Isoprinosine) - Polyribonucleoside (Ampligen)
- Ditiocarb sodium (Imuthiol) - Hydroxyurea
- Checkpoint Inhibitors
- Experimental vaccines - Investigational agents
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5.4.3 precautionary medications

The following medications may be used during the study with caution:
- Treatment with topical corticosteroids is allowed except at the proposed vaccination sites within 2 weeks prior to vaccination
- Use of local skin treatments is allowed, except in the targeted vaccination sites, within 2 weeks prior to vaccination. During the vaccination period, subjects should avoid manipulation of vaccinated areas to enable monitoring of possible vaccine-related toxicity. Sites may be rinsed with water, but soap and other possible irritants should be avoided. In the case of discomfort at the vaccination site, cold packs may be administered as needed. All other local treatments should only be administered after consultation with the investigator.
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5.5 dose modification guidelines and

A safety review team, internal to the sponsor, will regularly monitor all aspects of patient safety throughout this study. This team will be comprised of the medical monitor, a statistician, and ad hoc authorized representatives as appropriate. The safety review team will meet regularly to review all SAEs and will examine non-serious AEs, clinical laboratory data and other relevant safety data.
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5.5.1 local reactions

Local reactions, resulting from the vaccine will be graded using the local reaction assessment scale (Table 2). Table 2. Local Reaction Assessment Scale  
| Grade                            | Description                                                                                                |
|----------------------------------|------------------------------------------------------------------------------------------------------------|
| 0 = None                         | Not Applicable.                                                                                            |
| 1 = Mild                         | Macular or papular eruption, erythema, or induration that is  asymptomatic or mildly symptomatic.          |
| 2 = Moderate                     | Macular or papular eruption, erythema or induration with pruritus, or  other associated moderate symptoms. |
| 3 = Severe                       | Ulceration, superinfection, blistering, or phlebitis.                                                      |
| 4 = Potentially Life-Threatening | Necrosis of the skin.                                                                                      |  
If the subject develops a potentially life-threatening (Grade 4) local reaction thought to be probably or definitely related to vaccination, the safety review team must be notified within 24 hours. The safety review team will determine whether local reactions are due to the vaccine.
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5.5.2 systemic reactions

Systemic reactions will be graded according to the World Allergy Organization Subcutaneous Immunotherapy Systemic Reaction Grading System (Cox 2010) (Appendix B). The safety review team should be contacted within 48 hours for any non-local Grade 3 or 4 reactions (i.e., elevated temperature following vaccinations) thought probably or definitely related to vaccination. The safety review team will determine whether systemic reactions are due to the vaccine.
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5.5.3 anticancer drugs

Unanticipated and anticipated toxicities from the anticancer maintenance therapy regimen will be graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 (https://ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm). Any Grade 3 or 4 toxicities that result in a temporary or permanent change in an anticancer therapy must be reported to the safety review team within 4 days of knowledge of the change. Anticipated toxicities resulting from components of the anticancer maintenance therapy regimen will be managed by the subject's clinician according to best clinical practice including dose reductions when indicated. Subjects who change and/or permanently discontinue their anticancer maintenance therapy will be handled on a case-by-case basis through discussions with the safety review team to decide whether they can remain on study treatment/on study.
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5.5.4 dose limiting toxicity (dlt)

DLT is defined as any of the following occurring any time from Day 1 (Visit 2) until 21 days after administration of the vaccine (according to NCI CTCAE version 4.0, (https://ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm):
Hematologic  
- Grade 4 hematologic toxicity which lasts at least 5 days
- Grade 3 or higher thrombocytopenia which lasts at least 5 days
Non-hematologic:  
- Grade 4 or greater non-hematological local (at the site of vaccine injection) adverse events
- Grade 3 or greater non-hematological systemic adverse events with the exception of alopecia, anorexia, nausea, vomiting and fatigue, constipation and dehydration
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6 description of study assessments

The timing of the study assessments and procedures are summarized in the SoA in Appendix A. The investigator and sponsor will conduct the study in accordance with International Council for Harmonisation (ICH) Good Clinical Practice (GCP) and local regulations. Adherence to the study design requirements, including those specified in the SoA, is essential and required for study conduct, and the investigator must ensure that study procedures are performed as described in the protocol. During the study, procedures and observations will be monitored to confirm that study requirements are being followed as outlined in the SoA.
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6.1 informed consent form

Any patient who provides informed consent (i.e., signs and dates the informed consent form and assent form, if applicable) is considered a subject in the study. Subjects presently on study who have signed an informed consent form for a single vaccination will be asked to sign a new informed consent form for multiple (N=3) vaccinations.
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6.2 subject identification code

The following series of numbers will comprise the SIC: protocol identifier (e.g., 090701) to be provided by the sponsor, 2- or 3-digit study site number (e.g., 02) to be provided by the sponsor, and 3- or 4-digit subject number (e.g., 0003) reflecting the order of providing informed consent. For example, the third subject who signed an informed consent form at the site 02 will be identified as Subject 090701-020003. All study documents (e.g., CRFs, clinical documentation, sample containers, drug accountability logs, etc.) will be identified with the SIC. Additionally, a uniquely coded SIC(s) is permitted if it does not contain a combination of information that allows identification of a subject (e.g., collection of a subject's initials and birth date would not be permitted), in compliance with laws governing data privacy.
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6.3 screening log

The study site is responsible for maintaining a Screening log that includes all subjects who provided informed consent. The log also will serve to document the reason for screening failure. All screening data will be collected and reported in CRFs, regardless of screening outcome. If a subject is re-screened, the End of Study CRF should be completed, and a new informed consent form (ICF), new SIC and new CRF are required for that subject.
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6.4 vaccine site evaluation

A vaccine site evaluation for local and/or systemic adverse reactions will be performed in the clinic 1 hour after vaccination. If there are no adverse reactions noted within 1 hour after vaccination, subjects may be released from the clinic. Results of the site evaluation and toxicity grade, if appropriate, must be recorded in the source documents and documented on the CRF. For subsequent visits, vaccine site evaluation must be repeated. The following vaccination steps must be recorded in the source documents and documented on the CRF:  
- Vaccination sites
- Disinfection of vaccination sites
- Time of application