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6.5 evaluation of response to

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)
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6.6 immunogenicity and efficacy assessments

The following immunogenicity and efficacy parameters will be assessed:  
- PEPI Test
- Effector T cell response
- Memory T cell response
- TILs assessment - CT assessment
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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 sent to LabCorp for HLA genotyping. LabCorp will send the HLA genotype data to Treos 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.
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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. ELISPOT 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).
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6.6.3 memory t cell response

Memory T cells are a subset of antigen specific T cells that previously 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. During 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 by IFN-γ ELISPOT. It has been previously demonstrated that the PHPC assay identifies expandable memory T cells that can reveal previous exposure to specific peptide antigens (Calarota 2008).
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6.6.4 tumor infiltrating lymphocyte (tils)

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 reading 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 cancer 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).
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6.6.5 ct scan and recist/

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 evaluated 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.
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6.7 safety assessments 6.7.1 adverse

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. Patients should be monitored for AEs consistent with the current Investigator's Brochure for PolyPEPI1018.
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6.7.2 urgent safety measures

An urgent safety measure is an immediate action taken, which is not defined by the protocol, to protect subjects participating in a clinical trial from immediate harm. Urgent safety measures may be taken by the by the investigator or the safety review team, and may include any of the following:
1. Immediate change in study design or study procedures  
2. Temporary or permanent halt of a given clinical trial or trials 3. Any other immediate action taken to protect clinical trial participants from immediate hazard to their health and safety
The investigator or the safety review team may take appropriate urgent safety measures to protect subjects against any immediate hazard to their health or safety. The measures should be taken immediately and may be taken without prior authorization from the sponsor. In the event(s) of an apparent immediate hazard to the subject, the investigator will notify the medical monitor immediately by phone and confirm notification to the safety review team in writing as soon as possible, but within 1 calendar day after the change is implemented. The sponsor will also ensure the responsible ethics committee(s) and relevant competent authority(s) are notified of the urgent safety measures taken in such cases according to local regulations.
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6.7.3 untoward medical occurrences

Untoward medical occurrences occurring before the first exposure to IP are not considered AEs (according to the definition of AE, see Section 8). However, each serious untoward medical occurrence experienced before the IP exposure (i.e., from the time of signed informed consent up to but not including the IP exposure) will be described on the AE CRF and on the SAE Report Form. These events will not be considered as SAEs and will not be included in the analysis of SAEs. For the purposes of this study, each non-serious untoward medical occurrence experienced by a subject undergoing study-related procedure(s) before the IP exposure will be recorded on the AE CRF; these events will not be considered as AEs and will not be included in the analysis of AEs.
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6.7.4 non-medical complaints

A non-medical complaint (NMC) is any alleged product deficiency that relates to identity, quality, durability, reliability, safety, and performance of the product but did not result in an AE. NMCs include but are not limited to the following:  
1. A failure of a product to exhibit its expected pharmacological activity and/or design function, e.g. reconstitution difficulty 2. Missing components 3. Damage to the product or unit carton 4. A mislabeled product (e.g., potential counterfeiting/tampering) 5. A bacteriological, chemical, or physical change or deterioration of the product causing it to malfunction or to present a hazard or fail to meet label claims
Any NMCs of the product will be documented on an NMC form and reported to the sponsor within 1 business day. If requested, defective product(s) will be returned to the sponsor for inspection and analysis according to procedures.
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6.7.5 medical medication and non-drug

At Screening, the subject's medical history will be described for the following body systems including severity (defined in Section 8.2.1) or surgery and start and end dates, if known: eyes, ears, nose, and throat; respiratory; cardiovascular; gastrointestinal; musculoskeletal; neurological; endocrine; hematopoietic/lymphatic; dermatological; and genitourinary. All medications taken and non-drug therapies received from 3 months prior to enrollment until completion/termination will be recorded on the concomitant medications and non-drug therapies CRFs.
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6.7.6 physical examinations

Complete and targeted physical examinations will be performed as summarized in Appendix A. Complete physical examinations will be performed on the following body systems: general appearance, signs and symptoms (including, but not limited to the head and neck, eyes and ears, nose and throat, chest, lungs, heart, abdomen, extremities and joints, lymph nodes, skin, and neurological). Moreover, diagnoses, Karnofsky score, height, weight and vital signs will be registered. Finally, skin observation will be performed. At Screening, if an abnormal condition is detected, the condition will be described on the medical history CRF. At study visits, if a new abnormal or worsened abnormal pre-existing condition is detected, the condition will be described on the AE CRF. If the abnormal value was not deemed an AE because it was due to an error, due to a preexisting disease (described in Section 8.1.6), not clinically significant, a symptom of a new/worsened condition already recorded as an AE, or due to another issue that will be specified, the investigator will record the justification on the source record. At Visits 3 and 5, a targeted physical examination including vital signs should be driven by any new or previously identified signs and symptoms that the subject has experienced since the last visit.
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6.7.7 vital signs

Vital sign values (including seated blood pressure, pulse, respirations, and oral temperature) are to be measured at every visit as part of the complete and targeted physical examinations, and recorded on the CRF. At Day 1, measurement of vital signs must be performed in two occasions: before vaccination and 1 hour after last vaccination. For each abnormal vital sign value, the investigator will determine whether to report an AE (see definition in Section 8.1.1 and record the medical diagnosis [preferably], symptom, or sign on the AE CRF) to the safety review team. Additional tests and other evaluations required to establish the significance or etiology of an abnormal value, or to monitor the course of an AE should be obtained when clinically indicated. Any abnormal value that persists should be followed at the discretion of the investigator.
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6.7.8 clinical laboratory parameters 6.7.8.1

The hematology panel will consist of complete blood count (hemoglobin, hematocrit, erythrocytes [i.e., red blood cell count], and leukocytes [i.e., white blood cell count]) with differential (i.e., basophils, eosinophils, lymphocytes, monocytes, neutrophils) and platelet counts. The clinical chemistry panel will consist of sodium, potassium, chloride, bicarbonate, protein, albumin, ALAT, ASAT, gamma-glutamyl transpeptidase, total bilirubin, uric acid, blood urea nitrogen, creatinine, lactate dehydrogenase, creatine phosphokinase, alkaline phosphatase and glucose. Urinalysis will include description of color and clarity, specific gravity, pH, content of protein, glucose, nitrites and ketones. A microscopic analysis will be performed to check for red or white blood cells, casts, crystals, bacteria, yeast cells, parasites, and squamous cells. Urine and blood samples will be obtained for assessment of hematology and clinical chemistry at Screening, at Visit 2 (Day 1, Week 0), Visit 4 (Week 6), Visit 6 (Week 12), Visit 9 (Week 19), Visit 11 (Week 25), Visit 14 (Week 32) and Visit 16 (Week 38). Hematology and clinical chemistry assessments will be performed at the local laboratory at the investigator site.
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6.7.8.2 tumor biomarkers

Blood samples will be collected to evaluate tumor biomarkers, including carcinoembryonic antigen (CEA) and cancer antigen (CA 19-9).
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6.7.8.3 assessment of laboratory values

The investigator's assessment of each laboratory value will be recorded on the CRF. For each abnormal laboratory value, the investigator will determine whether the value is considered clinically significant or not. For clinically significant values, the investigator will indicate if the value constitutes a new AE (see definition in Section 8.1.1, and record the sign, symptom, or medical diagnosis on the AE CRF), is a symptom or related to a previously recorded AE, is due to a pre-existing disease (described in Section 8.2.2), or is due to another issue that will be specified. If the abnormal value was not clinically significant, the investigator will indicate the reason, i.e., because it is due to a preexisting disease, due to a lab error, or due to another issue that will be specified. Additional tests and other evaluations required to establish the significance or etiology of an abnormal value, or to monitor the course of an AE should be obtained when clinically indicated. Any abnormal value that persists should be followed at the discretion of the investigator. The investigator will communicate the AE to the safety review team for final evaluation.
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6.7.9 patient diary

A subject diary will be distributed to subjects on Day 1 (Visit 2), Day 92 ±2 (Visit 7) and Day 183 ±2 (Visit 12) to collect local signs related to the injection sites, physical fatigue, and body temperature during the first two weeks after the injection. Temperature should be consistently measured at the same location (e.g. underarm, ear, mouth etc.).
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6.8 procedures for monitoring subject

All study procedures are to be performed under the direct supervision of the investigator/a licensed healthcare professional at the study site, and thus, no separate procedures will be used to monitor subject compliance.
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7 assessments and procedures by

The timing of the study assessments and procedures are summarized in the SoA (Appendix A). Note: At the end of the follow up period (that is 12 weeks after each vaccination), ideally one week rest period should be observed before the next vaccination, as described in the SoA. However, up to 1 month rest period is allowed.
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7.1 screening period (visit 1

The following procedures will be performed at the Screening Visit:
- Informed consent
- Inclusion/exclusion criteria
- ECOG performance status
- Medical history - Medication history
- Complete physical exam
- Evaluation of response to first-line therapy - Cancer biopsy for TIL analysis
- CT and RECIST assessment
- Hematology and blood chemistries - Tumor biomarkers
- Urinalysis
- Urine pregnancy test - Blood collection for immunogenicity assays
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7.2 polypepi1018 administration (visit 2

The following procedures will be performed at Visits 2, 7 and 12:
- Concomitant medications
- Complete physical exam - Vaccine site evaluation
- Buccal swab
- Vaccine administration and 1-hour post-vaccination observation
- Distribute diary  
- Hematology and blood chemistries (not on Visits 7 and 12)
- Tumor biomarkers (not on Visits 7 and 12)
- Urinalysis (not on Visits 7 and 12) - Urine Pregnancy Test (not on Visits 7 and 12)
- Blood Collection for Immunogenicity Assays (not on Visits 7 and 12)
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7.3 follow-up period 7.3.1 visit

The following procedures will be performed at Visits 3, 5, 8, 10, 13 and 15:  
- Concomitant medications
- Targeted physical exam
- Vaccine site evaluation
- Collect diary
- Blood collection for immunogenicity assays
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7.3.2 visit 4 (day 43

The following procedures will be performed at Visits 4, 6, 9, 11, 14 and 16:  
- Concomitant medications - Complete physical exam
- Vaccine site evaluation - Collect diary
- CT and RECIST assessment (irRECIST assessment for subjects suspected of progression diseases at Visit 4)
- Hematology and blood chemistries
- Tumor biomarkers - Urinalysis
- Urine pregnancy test
- Blood collection for immunogenicity assays - Cancer biopsy for TIL analysis (only at Visit 6)
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7.4 last visit

Every effort will be made to have discontinued, withdrawn, and terminated subjects complete the assessments described for Visit 16 (see Section 7.3.2). If the Last Visit is performed as an unscheduled visit, the assessment results will be recorded with the Last Visit. If a subject terminates participation in the study and does not return for the Visit 16 assessments, their last recorded assessments shall remain recorded with their last visit. The reason for discontinuation will be recorded, and the data collected up to the time of discontinuation will be used in the analysis and included in the clinical study report. If additional assessments are required, the assessments shall be recorded separately. In the event of subject discontinuation due to an AE, clinical and/or laboratory investigations that are beyond the scope of the required study observations/assessments may be performed as part of the evaluation of the event. These investigations will take place under the direction of the investigator in consultation with the sponsor, and the details of the outcome may be reported to the appropriate regulatory authorities by the sponsor.
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8 adverse events and serious

Timely collection and assessment of SAE reports are critical for protecting the safety of patients. SAE reporting is a regulatory responsibility of the Sponsor under the Code of Federal Regulations (Title 21, Sub-Part B, section 312.32).
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8.1 definitions 8.1.1 adverse event

An AE is defined as any untoward medical occurrence in a subject administered an IP that does not necessarily have a causal relationship with the treatment. An AE can therefore be any unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom (e.g., rash, pain, discomfort, fever, dizziness, etc.), disease (e.g., peritonitis, bacteremia, etc.), or outcome of death temporally associated with the use of an IP, whether considered causally related to the IP. A treatment-emergent adverse event (TEAE) is defined as any event not present prior to the initiation of the treatments or any event already present that worsens in either intensity or frequency following exposure to the treatments.
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8.1.2 serious adverse event

An SAE is defined as an untoward medical occurrence that at any dose meets 1 or more of the following criteria:
1. Outcome is fatal/results in death (including fetal death).  
2. Is life-threatening - defined as an event in which the subject was, in the judgment of the safety review team, at risk of death at the time of the event; it does not refer to an event that hypothetically might have caused death had it been more severe.  
3. Requires inpatient hospitalization or results in prolongation of an existing hospitalization - inpatient hospitalization refers to any inpatient admission, regardless of length of stay.  
4. Results in persistent or significant disability/incapacity (i.e., a substantial disruption of a person's ability to conduct normal life functions).  
5. Is a medically important event - a medical event that may not be immediately life-threatening or result in death or require hospitalization but may jeopardize the subject or may require medical or surgical intervention to prevent one of the other outcomes listed in the definitions above.
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8.1.3 suspected unexpected serious adverse

Any suspected adverse reaction to study treatment (i.e., including active comparators) that is both serious and unexpected. The event(s) must meet all the following:
- Suspected adverse reaction
- Serious  
- Unexpected
- Assessed as related to study treatment
Once determined to meet the criteria for a SUSAR, an SAE should be submitted to regulatory agencies expeditiously.
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8.1.4 non-serious adverse event

A non-serious AE is an AE that does not meet the criteria of an SAE.
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8.1.5 unexpected adverse events

An unexpected adverse event is an AE whose nature, severity, specificity, or outcome is not consistent with the term, representation, or description used in the Reference Safety Information (RSI). "Unexpected" also refers to the AEs that are mentioned in the Investigator's Brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the product, but are not specifically mentioned as occurring with the product under investigation. The expectedness of AEs will be determined by the sponsor using the Investigator's Brochure as the RSI. This determination will include considerations such as the number of AEs previously observed, but not on the basis of what might be anticipated form the pharmacological properties of a product.
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8.1.6 preexisting diseases

Preexisting diseases that are present before entry in to the study are described in the medical history, and those that manifest with the same severity, frequency, or duration after IP exposure, will not be recorded as AEs. However, when there is an increase in the severity, duration, or frequency of a preexisting disease, the event must be described on the AE CRF.
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8.2 assessment of adverse events

Each AE from the IP exposure until study completion/discontinuation date will be described on the AE CRF using the medical diagnosis (preferred), or, if no diagnosis could be established at the time of reporting the AE, a symptom or sign, in standard medical terminology to avoid the use of vague, ambiguous, or colloquial expressions (see definition in Section 8.1). Each AE will be evaluated by the safety review team for:
1. Seriousness as defined in Section 8.1.2 2. Severity as defined in Section 8.2.1 3. Causal relationship to IP exposure or study procedure as defined in Section 8.2.2 For each AE, the outcome (i.e., recovering/resolving, recovered/resolved, recovered/resolved with sequelae, not recovered/not resolved, fatal, unknown) and if applicable (e.g. for maintenance therapy drugs) action taken (i.e., dose increased, dose not changed, dose reduced, drug interrupted, drug withdrawn, not applicable, or unknown) will also be recorded on the AE CRF. AEs attribution must be clearly specified, whether it is attributed to the vaccine or to maintenance drugs or any other concomitant medication. Recovering/resolving AEs will be followed until resolution, medically stabilized, or 30 days after the study completion, whichever comes first). If the severity rating for an ongoing AE changes before the event resolves, the original AE report will be revised (i.e., the event will not be reported as separate AE). During the course of any AE, the highest severity rating will be reported. Deviations from the protocol-specified dosage (including overdosing, under dosing, abuse, and withdrawal), treatment errors (including incorrect route of administration, use of an incorrect product, and deviations from the protocol-defined dosing schedule), failures of expected pharmacological actions, and unexpected therapeutic or clinical benefits will be followed with regard to occurrence of AEs, lack of efficacy, and/or other observations because these events may be reportable to regulatory authorities. Any pregnancy that occurs after administration of IP to a female subject or to a male subject whose partner gets pregnant during the study period will be reported on a Pregnancy Report Form and followed-up at estimated date of delivery. If an investigator becomes aware of an SAE occurring in a subject after study completion, the SAE must be reported on the provided SAE Report Form within 24 hours after awareness; no additional reporting on CRFs is necessary.
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8.2.1 severity

The safety review team will assess the severity of each AE using his/her clinical expertise and judgment based on the most appropriate description below (according to CTCAE Version 4.0):
1. Mild (Grade 1)  
o Asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not indicated 2. Moderate (Grade 2)
o Minimal, local or noninvasive intervention indicated; limiting age-appropriate instrumental activities of daily living (ADL)
3. Severe (Grade 3)
o Severe or medically significant but not immediately life-threatening; hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care ADL
4. Life threatening (Grade 4)
o Life-threatening consequences; urgent intervention indicated 5. Death related to AE (Grade 5)
o Grade 5 is not appropriate for some AEs and therefore is not an option These severity definitions will also be used to assess the severity of an AE with a study-related procedure(s), if necessary.
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8.2.2 causality

Causality is a determination of whether there is a reasonable possibility that the IP is etiologically related to/associated with the AE. Causality assessment includes, e.g., assessment of temporal relationships, dechallenge/rechallenge information, association (or lack of association) with underlying disease, presence (or absence) of a more likely cause, and physiological plausibility. For each AE, the safety review team will assess the causal relationship between the IP and the AE using his/her clinical expertise and judgment according to the following most appropriate algorithm for the circumstances of the AE:
1. Not related (both circumstances must be met)  
o Is due to underlying or concurrent illness, complications, concurrent treatments, or effects of concurrent drugs o Is not associated with the IP (i.e., does not follow a reasonable temporal relationship to the administration of IP or has a much more likely alternative etiology).  
2. Unlikely related (either 1 or both circumstances are met)
o Has little or no temporal relationship to the IP o A more likely alternative etiology exists 3. Possibly related (both circumstances must be met)
o Follows a reasonable temporal relationship to the administration of IP o An alternative etiology is equally or less likely compared to the potential relationship to the IP
4. Probably related (both circumstances must be met)
o Follows a strong temporal relationship to the administration of IP, which may include but is not limited to the following:
- Reappearance of a similar reaction upon re-administration (positive rechallenge)
- Positive results in a drug sensitivity test (skin test, etc.) - Toxic level of the IP as evidenced by measurement of the IP
concentrations in the blood or other bodily fluid o Another etiology is unlikely or significantly less likely 5. Definitely related o There is clear evidence to suggest a causal relationship and other possible contributing factors can be ruled out.
These causality definitions will also be used to assess the relationship of an AE with a study-related procedure(s), if necessary.
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9 statistics 9.1 analysis populations

The Safety Analysis Set (SAS) will include all subjects who receive at least 1 dose of the IP.  
This population will be the primary analysis set for safety outcomes.
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9.2 replacement of subjects

Any subject who withdraws from the study prematurely, or who discontinues study vaccination prematurely without experiencing a primary safety endpoint (Section 9.4) before the Day 85 (Week 12, Visit 6) evaluation, and whose reasons for withdrawing from the study, or discontinuing study vaccination administration, are unrelated to any real or perceived effect of the study vaccination or its administration, will be replaced with another subject. If possible, all subjects who discontinue study treatment prematurely will be followed for 12 weeks for all study evaluations. A maximum of 5 subjects will be replaced without asking the sponsor's permission. If more than 5 subjects need to be replaced, then the study team may, for this reason, ask the sponsor for approval.
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9.3 handling of missing unused

Only data collected from the visits by subjects will be included in the statistical analysis. No imputation will be carried out on missing values.
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9.3.2 intermediate missing data

Only data collected from the visits by subjects will be included in the statistical analysis. No imputation will be carried out on missing values.
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9.4 primary endpoint

The primary endpoint is the occurrence of at least 1 ≥Grade 4 local 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 (as judged by the safety review team, including site clinicians on the team) any time from Day 1 (Visit 2) until 21 days after administration of the vaccine (Visit 3).
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9.5 statistical analyses 9.5.1 safety

Major components for the safety analysis will include:  
- AEs solicited at every study visit, recorded, and coded according to the version of the Medical Dictionary for Regulatory Activities (MedDRA) that is current on the date of study initiation
- SAEs
- Vital signs and physical examination findings - Laboratory parameters
All safety assessments will be summarized in a descriptive manner similar to the analysis described in Section 9.5.1.1. Data reported by the subjects in Patient Diary, including local signs related to the injection sites, physical fatigue, and body temperature, will be summarized in a descriptive manner.
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9.5.1.1 analysis of adverse events

All AEs will be analyzed in terms of descriptive statistics and qualitative analysis. Adverse events will be listed for each subject and summarized by system organ class (SOC) and preferred term according to the latest version of MedDRA. In addition, summaries of AEs by severity and relationship to IP will be presented. All safety and tolerability data recorded during the study will be listed and summarized over time, as appropriate. For the purpose of summaries and listings, the durations of AEs will be calculated as follows: (stop [day] - start [day]) + 1 day, which yields the number of days on which the AE was present. Treatment emergent AEs are defined as AEs that first occurred or worsened in severity after the first administration of the IP. Adverse event summaries will include incidence of TEAEs, SAEs including deaths, AEs that led to IP discontinuation, AEs by maximum severity and relationship to IP, and temporally infusion related AEs. The version of the MedDRA that is current on the date of study initiation will be used throughout conduct and data analysis. All summaries will be presented by MedDRA SOC and preferred term for each treatment group. Safety/tolerability and AE data will be presented in individual listings.
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9.5.2 pepi and immunogenicity analyses

Each of the 6 peptides in PolyPEPI1018 contain 2 epitopes (for a total of 12 epitopes) selected to elicit a T cell response in a high portion of subjects. The PEPI Test identifies which epitope represents a PEPI (that is capable of inducing a T cell response) in each subject. A PEPI is unique and is based on the subject's HLA genotype. Therefore, a selected epitope encoded in PolyPEPI1018 might be a PEPI for 1 individual and not for others, and vice versa. A subject may have more than 1 PEPI, or no PEPI. The PEPI Test (described in Section 6.6.1) will be performed in each subject who is enrolled in the study from the results of the HLA genotyping. The ELISPOT assay (described in Section 6.6.2) and the PHPC assay (described in Section 6.6.3) measure effector and memory responses, respectively. These assays will be performed in each subject from blood samples collected during a time course from Baseline until the Last Visit. Based on the results of the PEPI Test and the immunogenicity tests (ELISPOT and PHPC) the secondary outcome measures will be analyzed as described in Section 3.2.2. Immunogenicity analyses may be performed at any time during the trial, however, the results of the PEPI Test will be obtained before the results of the corresponding immunogenicity tests are available. Final analysis will be based on all data up to Day 85 (Week 12, Visit 6) for all study subjects (data from the start of study treatment of the last accrued subject through Day 85 [Week 12, Visit 6]).
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9.5.3 efficacy analyses

Efficacy will be evaluated as 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. RECIST and irRECIST criteria will be used. Primary and secondary analyses will be summarized in a descriptive manner.
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9.5.4 exploratory analyses

- Pearson correlation coefficients will be calculated to measure correlations. The significance of the correlations will be tested using standard method of estimating significance of correlations (� −2 1−2 follows the t distribution with degree of freedom n-2, where r is the Pearson correlation and n is the number of measurements)
- Correlated data points will be plotted and trend lines will be obtained using perpendicular (orthogonal) regression, since both axes contain measurements and/or predicted values (PEPIs predicted by the PEPI Test)
- The significance of the changes will be tested by non-parametric tests (Wilcoxon signed-rank test). Non-parametric test is preferred due to the small sample size and the questionable normality of the data
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9.6 determination of sample size

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 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 4, which is generated from 10,000 simulations. The x-axis shows the number of positive tests, and the y-axis shows the probabilities.  
  
Figure 4. 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 5, the x-axis shows the number of subjects with a given probability and the y-axis shows the probability. Figure 5. Expected Distribution of Probabilities Having a Given Number of Subjects with Positive Immune Response for Two or More Antigens
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10 ethical and legal considerations

This study will be conducted in accordance with the protocol and with ICH GCP guidelines, as well as all applicable country and regional legal and regulatory requirements. The investigator is responsible for ensuring that this protocol, the site's ICF, and any other information that will be presented to potential subjects are reviewed and approved by the appropriate IRB or IEC prior to the enrollment of any study subjects.
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10.2 ethics committee and regulatory

Before patients participate in this study, the protocol, informed consent form, any promotional material/advertisements, and any other written information will be reviewed and approved/given favorable opinion by the Ethics Committee (EC) and applicable regulatory authorities. The Investigator's Brochure will be provided for review. The EC's composition or a statement that the EC's composition meets applicable regulatory criteria will be documented. The study will commence only upon the sponsor's receipt of approval/favorable opinion from the EC and, if required, upon the sponsor's notification of applicable regulatory authority(s) approval, as described in the Clinical Trial Agreement. If the protocol or any other information given to the subject is amended, the revised documents will be reviewed and approved/given favorable opinion by the EC and applicable regulatory authorities, where applicable. The protocol amendment will only be implemented upon the sponsor's receipt of approval and, if required, upon the sponsor's notification of applicable regulatory authority(s) approval.
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10.3 informed consent and human

Investigators will choose patients for participation while considering the study eligibility criteria. The investigator will exercise no selectivity so that no bias is introduced from this source. All patients and/or their legally authorized representative must sign an informed consent form before entering into the study according to applicable national and local regulatory requirements and ICH GCP. An assent form may be provided and should be signed by patients less than 18 years of age. Before use, the informed consent form will be reviewed by the sponsor and approved by the EC and regulatory authority(s), where applicable, (see Section 10.2). The informed consent form will include a comprehensive explanation of the proposed treatment without any exculpatory statements, in accordance with the elements required by ICH GCP and applicable national and local regulatory requirements. Patients or their legally authorized representative(s) will be allowed sufficient time to consider participation in the study. By signing the informed consent form, patients/healthy volunteers or their legally authorized representative(s) agree that they will complete all evaluations required by the study, unless they withdraw voluntarily or are terminated from the study for any reason. The sponsor will provide to the investigator in written form any new information that significantly bears on the subjects' risks associated with IP exposure. The informed consent will be updated, if necessary. This new information and/or revised informed consent form, which has been approved by the applicable EC and regulatory authorities, where applicable, will be provided by the investigator to the subjects who consented to participate in the study. 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.
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10.4 confidentiality

The investigator will comply with the confidentiality policy as described in the Clinical Trial Agreement.
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11 administrative consideration 11.1 investigator's

The investigator will comply with the protocol (which has been approved/given favorable opinion by the EC), ICH GCP, and applicable national and local regulatory requirements as described in the Clinical Trial Agreement. The investigator is ultimately responsible for the conduct of all aspects of the study at the study site and verifies by signature the integrity of all data transmitted to the sponsor. The term "investigator" as used in this protocol as well as in other study documents, refers to the investigator or authorized study personnel that the investigator has designated to perform certain duties. Sub-investigators or other authorized study personnel are eligible to sign for the investigator, except where the investigator's signature is specifically required. The coordinating investigator will be selected before study start. The coordinating investigator will sign the clinical study report.
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11.2 study training

The sponsor will ensure that the investigator and study site personnel understand all requirements of the protocol, the investigational status of the IP, and his/her regulatory responsibilities as an investigator. Training may be provided at an investigator's meeting, at the study site, and/or by instruction manuals. In addition, the sponsor will be available for consultation with the investigator and will serve as the liaison between the study site and the sponsor.
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11.3 study monitoring

The study monitor is responsible for ensuring and verifying that each study site conducts the study according to the protocol, standard operating procedures, other written instructions/agreements, ICH GCP, and applicable national and local regulatory guidelines/requirements. The investigator will permit the study monitor to visit the study site at appropriate intervals, as described in the Clinical Trial Agreement. Monitoring processes specific to the study will be described in the clinical monitoring plan.
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11.4 safety monitoring

Accrual, withdrawals from treatment, all toxicities, and AEs will be monitored during the study. Reports should be reviewed every 2 weeks by the safety review team from one week after the study opens until it closes. If 2 subjects experience a primary safety endpoint (Section 9.4), or 1 subject experiences a ≥Grade 4 AE that is probably or definitely related to study treatment, enrollment will be suspended. Depending on the severity of the AE, study treatment administration to subjects may be terminated, pending a subsequent decision by the study team on how to proceed. When the last subject reaches Week 12, the safety review team will review all available safety data, including hematology, chemistries, tumor biomarkers, viral load data, and all clinical events.
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11.5 audits

The sponsor and/or sponsor's representatives may conduct audits to evaluate study conduct and compliance with the protocol, standard operating procedures, other written instructions/agreements, ICH GCP, and applicable national and local regulatory guidelines/requirements. The investigator will permit auditors to visit the study site, as described in the Clinical Trial Agreement. Auditing processes specific to the study will be described in the audit plan.
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11.6 documentation 11.6.1 case report

The investigator will maintain complete and accurate paper format study documentation in a separate file. Study documentation may include information defined as "source data" (see Section 11.6.3), records detailing the progress of the study for each subject, signed informed consent forms, correspondence with the EC and the study monitor/sponsor, screening information, CRFs, SAE reports (SAERs), laboratory reports (if applicable), and data clarifications requested by the sponsor. The investigator will comply with the procedures for data recording and reporting. Any corrections to paper study documentation must be performed as follows: 1) the first entry will be crossed out entirely, remaining legible; and 2) each correction must be dated and initialed by the person correcting the entry; the use of correction fluid and erasing are prohibited. The investigator is responsible for the procurement of data and for the quality of data recorded on the CRFs. CRFs will be provided in electronic form. Only authorized study site personnel will record or change data on the CRFs. If data is not entered on the CRFs during the study visit, the data will be recorded on paper, and this documentation will be considered source documentation. Changes to a CRF will require documentation of the reason for each change. An identical (electronic/paper) version of the complete set of CRFs for each subject will remain in the investigator file at the study site in accordance with the data retention policy (see Section 11.6.4). The handling of data by the sponsor, including data quality assurance, will comply with regulatory guidelines (e.g., ICH GCP) and the standard operating procedures of the sponsor. Data management and control processes specific to the study will be described in the data management plan.
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11.6.2 direct access to source

The investigator/study site will cooperate and provide direct access to study documents and data, including source documentation for monitoring by the study monitor, audits by the sponsor or sponsor's representatives, review by the IEC, and inspections by applicable regulatory authorities, as described in the Clinical Trial Agreement. If contacted by an applicable regulatory authority, the investigator will notify the sponsor of contact, cooperate with the authority, provide the sponsor with copies of all documents received from the authority, and allow the sponsor to comment on any responses, as described in the Clinical Trial Agreement.
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11.6.3 source data

Per ICH GCP, source data are defined as all information in original records and certified copies of original records of clinical findings, observations, or other activities in a clinical trial that are necessary for the reconstruction and evaluation of the trial. Source data are contained in source documents (original records or certified copies), which may be in paper and/or electronic format. Source data for this study comprise the following: hospital records, medical records, clinical and office charts, laboratory notes, memoranda, subjects' diaries or evaluation checklists, outcomes reported by subjects, pharmacy dispensing records, recorded data from automated instruments, copies or transcriptions certified after verification as being accurate copies, microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject files, and records kept at the pharmacy, at the laboratories and at medico-technical departments involved in the clinical study. No data will be entered directly onto the CRF. For additional information on study documentation and CRFs, see Section 11.6.1.
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11.6.4 document and data retention

The investigator will retain study documentation and data (paper and electronic forms) in accordance with applicable regulatory requirements and the document and data retention policy, as described in the Clinical Trial Agreement.
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11.7 non-compliance with the protocol

The investigator may deviate from the protocol only to eliminate an apparent immediate hazard to the subject. In the event(s) of an apparent immediate hazard to the subject, the investigator will notify the sponsor immediately by phone and confirm notification to the sponsor in writing as soon as possible, but within 1 calendar day after the change is implemented. The sponsor will also ensure the responsible EC and relevant competent authority is notified of the urgent measures taken in such cases according to local regulations. If monitoring and/or auditing identify serious and/or persistent non-compliance with the protocol, the sponsor may terminate the investigator's participation. The sponsor will notify the EC and applicable regulatory authorities of any investigator termination.
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11.8 financing and insurance

The investigator will comply with investigator financing, investigator/sponsor insurance, and subject compensation policies, if applicable, as described in the Clinical Trial Agreement.
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11.9 publication policy

The investigator will comply with the publication policy as described in the Clinical Trial Agreement.
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12 references

Bever KM and Le DT. An Expanding Role for Immunotherapy in Colorectal Cancer. J Natl Compr Canc Netw. 2017;15(3):401-410. Calarota SA, Foli A, Maserati R, et al. HIV-1-specific T cell precursors with high proliferative capacity correlate with low viremia and high CD4 counts in untreated individuals. J Immunol. 2008;180(9):5907-5915. Cassidy S and Syed BA. Colorectal cancer drugs market. Nat Rev Drug Discov. 2017; 16(8):525-526. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39(1):1-10. Cox L, Larenas-Linnemann D, Lockey RF, et al. Speaking the same language: The World Allergy Organization Subcutaneous Immunotherapy Systemic Reaction Grading System. Allergy Clin Immunol. 2010;125(3):569-74. Cremolini C, Loupakis F, Antoniotti C, et al. FOLFOXIRI plus bevacizumab versus FOLFIRI plus bevacizumab as first-line treatment of patients with metastatic colorectal cancer: updated overall survival and molecular subgroup analyses of the open-label, phase 3 TRIBE study. Lancet Oncol. 2015;16(13):1306-15. DeNardo DG and Coussens LM. Inflammation and breast cancer. Balancing immune response: crosstalk between adaptive and innate immune cells during breast cancer progression. Breast Cancer Res. 2007;9(4):212. Emens LA and Middleton G. The interplay of immunotherapy and chemotherapy: harnessing potential synergies. Cancer Immunol Res. 2015;3(5):436-43. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E35986. Heinemann V, von Weikersthal LF, Decker T, et al. FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as first-line treatment for patients with metastatic colorectal cancer (FIRE-3): a randomised, open-label, phase 3 trial. Lancet Oncol. 2014;15(10):106575. Grothey A, Van Cutsem E, Sobrero A, et al. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet. 2013;381(9863):303–312. Klintrup K, Makinen JM, Kauppila S, et al. Inflammation and prognosis in colorectal cancer. Eur J Cancer. 2005;41(17):2645-2654. Loupakis F, Cremolini C, Masi G, et al. Initial Therapy with FOLFOXIRI and Bevacizumab for Metastatic Colorectal Cancer. N Engl J Med. 2014;371(17):1609-18. Mansfield AS, Nevala WK, Lieser EAT, et al. The immunomodulatory effects of bevacizumab on systemic immunity in patients with metastatic melanoma. Oncoimmunology. 2013;2(5):e24436.  
Mariotto AB, Yabroff KR, Shao, et al. Projections of the cost of cancer care in the United States: 2010-2020. Journal of the National Cancer Institute. 2011;103(2):117–128. Ott PA, Hu Z, Keskin DB, et al. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature. 2017;547:217–221. Sahin U, Derhovanessian E, Miller M, et al. Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature. 2017;547:222–226. Saltz LB. Clarke S, Diaz-Rubio E, et al. Bevacizumab in combination with oxaliplatin-based chemotherapy as first-line therapy in metastatic colorectal cancer: a randomized phase III study. J Clin Oncol. 2008;26(12):2013-9. Schmidt M, Bohm D, von Torne C, et al. The humoral immune system has a key prognostic impact in node-negative breast cancer. Cancer Res. 2008;68(13):5405–13. Seymour MT, Brown SR, Middleton G, et al. Panitumumab and irinotecan versus irinotecan alone for patients with KRAS wild-type, fluorouracil-resistant advanced colorectal cancer (PICCOLO): a prospectively stratified randomised trial. The Lancet Oncology. 2013;14(8):749–759. Terme M, Pernot S, Marcheteau E, et al. VEGFA-VEGFR Pathway Blockade Inhibits Tumor-Induced Regulatory T-cell Proliferation in Colorectal Cancer. Cancer Res. 2013; 72(2):539-49. Venook AP, Niedzwiecki D, Lenz HJ, et al. Effect of First-Line Chemotherapy Combined With Cetuximab or Bevacizumab on Overall Survival in Patients With KRAS Wild-Type Advanced or Metastatic Colorectal Cancer. JAMA. 2017;317(23):2392-2401. Walter S, Weinschenk T, Stenzi A, Zdrojowy R. Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival.  
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| Period                                                                  | Screening   | Vaccination and Evaluation   |       |       |      |      |
|-------------------------------------------------------------------------|-------------|------------------------------|-------|-------|------|------|
| Visit Number                                                            | 1           | 2                            | 3     | 4     | 5    | 6    |
| End of Week                                                             | -3 to -1    | 0                            | 3     | 6     | 9    | 12   |
| Day                                                                     | -21 to -1   | 1                            | 22 ±2 | 43 ±2 | 64±2 | 85±2 |
| Informed Consent                                                        | X           |                              |       |       |      |      |
| Inclusion/Exclusion Criteria                                            | X           |                              |       |       |      |      |
| ECOG Performance Status                                                 | X           |                              |       |       |      |      |
| Medical History                                                         | X           |                              |       |       |      |      |
| Medication History                                                      | X           |                              |       |       |      |      |
| Concomitant Medications                                                 | X           | X                            | X     | X     | X    |      |
| Complete Physical Exam                                                  | X           | X                            | X     | X     |      |      |
| Targeted Physical Exam                                                  | X           | X                            |       |       |      |      |
| Vital Signs                                                             | X           | X                            | X     | X     | X    | X    |
| Vaccine Site Evaluation                                                 | X           | X                            | X     | X     | X    |      |
| Evaluation of Response to First-line Therapy                            | X           |                              |       |       |      |      |
| Buccal Swab                                                             | X           |                              |       |       |      |      |
| Vaccine Administration/ 1-hour Observation                              | X           |                              |       |       |      |      |
| Distribute Diary1                                                       | X           |                              |       |       |      |      |
| Collect Diary                                                           | X           |                              |       |       |      |      |
| Cancer Biopsy/ Tumor Infiltrating Lymphocytes  Analysis                 | X           | X                            |       |       |      |      |
| CT and RECIST (irRECIST) Assessment                                     | X           | X                            | X     |       |      |      |
| Hematology & Blood Chemistries                                          | X           | X                            | X     | X     |      |      |
| Tumor Biomarkers                                                        | X           | X                            | X     | X     |      |      |
| Urinalysis                                                              | X           | X                            | X     | X     |      |      |
| Urine Pregnancy Test                                                    | X           | X                            | X     | X     |      |      |
| Blood Collection for PBMC and Immunogenicity  Assays (ELISpot and PHPC) | X           | X                            | X     | X     | X    | X    |
| Assessment                                                              |             |                              |       |       |      |      |  
Abbreviations: ECOG = Eastern Cooperative Oncology Group; CT = computerized tomography 1. The Patient Diary will be used to collect local signs related to the injection sites, physical fatigue, and body temperature  
| Period                                                                  | Vaccination and Evaluation   |        |        |        |        |
|-------------------------------------------------------------------------|------------------------------|--------|--------|--------|--------|
| Visit Number                                                            | 7                            | 8      | 9      | 10     | 11     |
| End of Week                                                             | 13                           | 16     | 19     | 22     | 25     |
| Day                                                                     | 92 ±2                        | 113 ±2 | 134 ±2 | 155 ±2 | 176 ±2 |
| Informed Consent for additional vaccinations2                           | X                            |        |        |        |        |
| Concomitant Medications                                                 | X                            | X      | X      | X      | X      |
| Complete Physical Exam                                                  | X                            | X      | X      |        |        |
| Targeted Physical Exam                                                  | X                            | X      |        |        |        |
| Vital Signs                                                             | X                            | X      | X      | X      | X      |
| Vaccine Site Evaluation                                                 | X                            | X      | X      | X      | X      |
| Vaccine Administration/ 1-hour Observation                              | X                            |        |        |        |        |
| Distribute Diary1                                                       | X                            |        |        |        |        |
| Collect Diary                                                           | X                            |        |        |        |        |
| Cancer Biopsy/ Tumor Infiltrating Lymphocytes  Analysis                 | X                            |        |        |        |        |
| CT and RECIST (irRECIST) Assessment                                     | X                            | X      |        |        |        |
| Hematology & Blood Chemistries                                          | X                            | X      |        |        |        |
| Tumor Biomarkers                                                        | X                            | X      |        |        |        |
| Urinalysis                                                              | X                            | X      |        |        |        |
| Urine Pregnancy Test                                                    | X                            | X      |        |        |        |
| Blood Collection for PBMC and Immunogenicity  Assays (ELISpot and PHPC) | X                            | X      | X      | X      |        |
| Abbreviations: CT = computerized tomography Assessment                  |                              |        |        |        |        |  
| Period                                                                  | Vaccination and Evaluation   |        |        |        |        |
|-------------------------------------------------------------------------|------------------------------|--------|--------|--------|--------|
| Visit Number                                                            | 12                           | 13     | 14     | 15     | 16     |
| End of Week                                                             | 26                           | 29     | 32     | 35     | 38     |
| Day                                                                     | 183 ±2                       | 204 ±2 | 225 ±2 | 246 ±2 | 267 ±2 |
| Concomitant Medications                                                 | X                            | X      | X      | X      | X      |
| Complete Physical Exam                                                  | X                            | X      | X      |        |        |
| Targeted Physical Exam                                                  | X                            | X      |        |        |        |
| Vital Signs                                                             | X                            | X      | X      | X      | X      |
| Vaccine Site Evaluation                                                 | X                            | X      | X      | X      | X      |
| Vaccine Administration/ 1-hour Observation                              | X                            |        |        |        |        |
| Distribute Diary1                                                       | X                            |        |        |        |        |
| Collect Diary                                                           | X                            |        |        |        |        |
| Cancer Biopsy/ Tumor Infiltrating Lymphocytes  Analysis                 | X                            |        |        |        |        |
| CT and RECIST (irRECIST) Assessment                                     | X                            | X      |        |        |        |
| Hematology & Blood Chemistries                                          | X                            | X      |        |        |        |
| Tumor Biomarkers                                                        | X                            | X      |        |        |        |
| Urinalysis                                                              | X                            | X      |        |        |        |
| Urine Pregnancy Test                                                    | X                            | X      |        |        |        |
| Blood Collection for PBMC and Immunogenicity  Assays (ELISpot and PHPC) | X                            | X      | X      | X      |        |
| Abbreviations: CT = computerized tomography Assessment                  |                              |        |        |        |        |  
Note: At the end of the follow up period (that is 12 weeks after each vaccination), ideally one week rest period should be observed before the next vaccination, as described in the above SoA. However, up to 1 month rest period is allowed.  
APPENDIX B. THE WORLD ALLERGY **ORGANIZATION SUBCUTANEOUS IMMUNOTHERAPY SYSTEMIC**
REACTION GRADING SYSTEM
See attached Article (13 pages) by Cox L. et al