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 th at study requirements are being followed as outlined in the SoA. 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. 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 i t 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. 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 Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 47 of 81 CONFIDENTIAL failure. All screenin g 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. 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 6.5 Evaluation of Response to First- line Therapy During the Screening Period, results from a CT scan which was performed at the completion of the first -line treatment regimen must be obtained (i.e., eligibility CT scan). The results will be evaluated by the investigator and must confirm that the subject has responded to first- line chemotherapy with documentation of cancer regression or lack of progression, according to RECIST version 1.1 criteria (https://ctep.cancer.gov/protocolDevelopment/docs/recist_guideline.pdf ) 6.6 Immunogenicity and Efficacy Assessments The following immu nogenicity and efficacy parameters will be assessed: • PEPI Test • Effector T cell response • Memory T cell response Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 48 of 81 CONFIDENTIAL • TILs assessment • CT assessment 6.6.1 PEPI Test To identify PEPIs the sponsor developed a novel PEPI Test. 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 PEPI Test will be used to identify PEPIs among the 12 selected epitopes of PolyPEPI1018 in each study subject . To perform the PEPI Test a buccal swab sample will be collected on Day 1 (Visit 2) and sen t to LabCorp for HLA genotyping. LabCorp will send the HLA genotype data to T reos Bio ZRT (sponsor) to perform the PEPI Test. The sponsor will perform the PEPI Test before any immunology or efficacy assessment becomes available, essentially in a blinded fashion. The PEPI Test results will be unblinded and the data compared with immunology and efficacy results as described in Section 9.5.2. 6.6.2 Effector T Cell Response Effector T cells are generated through activation of naïve or memory T cells following recognition of epitopes presented by the HLA molecules. E LISPOT assay is a routine test to be utilized for the detection of effector T cells that respond to PolyPEPI1018 treatment from a population of peripheral blood mononuclear cells (PBMCs). 6.6.3 Memory T Cell Response Memory T cells are a subset of antigen specific T cells that previ ously encountered and responded to their cognate epitope. Such T cells are primed by HLA- restricted peptides and originate either from cancer cells or from the vaccine. Durin g a second activation, memory T cells can proliferate faster than during priming. This behavior is captured by the PHPC assay, abbreviated from “T cell precursors with high proliferation capacity” that allows the expansion of memory cells during prolonged culture and then detects the progeny of the antigen-specific memory T cell clone b y IFN-γ ELISPOT. It has been previously Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 49 of 81 CONFIDENTIAL demonstrated that the PHPC assay identifies expandable memory T cells that can reveal previous exposur e to specific peptide antigens ( Calarota 2008). 6.6.4 Tumor Infiltrating Lymphocyte (TIL s) The presence of TILs is associated with favorable long ‐term outcome in several cancers, including breast cancer and CRC ( DeNardo 2007; Schmidt 2008; Klintrup 2005). During the procedure tumor specimens are fixed in 10% formalin, embedded in paraffin, and 4‐μm ‐thick sections are prepared for hematoxylin and eosin staining or other appropriate staining and immunohistochemistry (IHC). All stainings and data readin g are performed and reviewed by the same pathologist. The unused tumor sections will be retained according to the site procedure. TILs are assessed according to published criteria (Klintrup 2005) using a 4- degree scale at the deepest area of the invasive margin. A score of 0 is given when there is no increase in inflammatory cells, 1 denotes a mild and patchy increase in inflammatory cells, 2 denotes a moderate and band like inflammatory infiltrate with some destruction of ca ncer cell islands, and 3 denotes a marked and florid cuplike inflammatory infiltrate with frequent destruction of cancer cell islands. These scores can be subsequently classified as low grade (scores 0 and 1) and high grade (scores 2 and 3). 6.6.5 CT scan and RECIST/ irRECIST evaluation CT scan will be performed at the site and original data will be retained either in a CD -ROM format or in a data room. RECIST criteria will be evaluate d at baseline, week 6 (Visit 4), week 12 (Visit 6), week 25 (Visit 11), week 38 (Visit 16) based on the CT scan results. If progressive disease (PD) is suspected at week 6, irRECIST evaluation (https://www.parexel.com/files/7914/2186/7838/irrecist- path-PDF.pdf) will be implemented; if the subject remains in the study, the CT scan on Wk 12 will be evaluated according to irRECIST criteria. Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 50 of 81 CONFIDENTIAL 6.7 Safety Assessments 6.7.1 Adverse Events Details on definitions and reporting of AEs are provided in Section 8. All AEs will be recorded from the time of signing of the ICF until completion of the Last Visit.