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 m CRC (Loupakis 2014). Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 19 of 81 CONFIDENTIAL Despite the avail able treatment options, r esponse 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 dis ease, 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). 1.2 PolyPEPI 1018 and Companion Diagnostic Vaccine Overview PolyPEPI 1018 CRC Vaccine (PolyPEPI 1018) 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 wh o most likely respond to vaccine treatment. The CDx uses th e 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 Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 20 of 81 CONFIDENTIAL PolyPEPI10108 CDx will use the PEPI T est to identify the patient’s PEPIs from PolyPEPI1018. D ata 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. Table 1. Synthetic Peptide Sequence s 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 PolyPEPI 1018, 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 PolyPEPI 1018, tumor biopsy and CTA expression analysis, a risky, inaccurate and costly procedure1, is not required prior to vaccination with PolyPEPI 1018. 1 There is no validated diagnostic device available to perform tumor antigen expression profiling from tumor biopsies. In addition, a biopsy does not comprehensively characterize a heterogeneous tumor, especially in the metastatic stage . Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 21 of 81 CONFIDENTIAL Figure 1. Cancer Testis Antigens Targeted by PolyPEPI1018 Abbreviations: CRC = c olorectal 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. 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. PolyPEPI 1018 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). Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 22 of 81 CONFIDENTIAL Figure 2. Mechanism of Action of Precision PolyPEPI1018 CRC Vaccin e 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: PolyPEPI101 8 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. 1.2.2 Preclinical and Clinical Background for PolyPEPI 1018 PolyPEPI 1018 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. 1.2.2.1 Safety of P eptide Vaccines and MontanideTM 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 Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 23 of 81 CONFIDENTIAL 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, p atients 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 immu ne 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 contain ing 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, colit is, rectal bleeding and bladder-vaginal fistulae. 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, t he main limitation of these peptide vaccines is the absence of the induction of CTL responses against multiple antigens expressed in tumor cells. Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 24 of 81 CONFIDENTIAL 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 polyva lent 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. 1.3 Rationale for Study Design and Dose Regimen 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 o ptimal 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. Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 25 of 81 CONFIDENTIAL Moreover, a clean readout of immune res ponses 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 S creening since a robust immune system is needed to elicit an appropriate immune res ponse to vaccination with PolyPEPI1018. Also for this reason, s ubjects must have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. The sponsor intends to explore whether multiple vaccination s 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 vaccination s. Consequent ly, 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 (5- fluorouracil or capecitabine) plus the same biologic agent (bevacizumab, cetuximab or panitumumab) used during induction. I t is typically assumed that chemotherapy is immune Treos Bio ZRT OBERTO -101 Amendment 3 Product: PolyPEPI 1018 Colorectal Cancer Vaccine Page 26 of 81 CONFIDENTIAL 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. 1.4 Benefits a nd Risks It is expected that PolyPEPI 1018 will induce PEPI-specific immune responses against multiple CTAs expressed in CRC cells. Previous clinical studies have demonstrated that peptide vaccines similar to PolyPEPI 1018 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.