........................ 74 13.1 APPENDIX A: PERFORMANCE STATUS CRITERIA ................................ ................................ .74 13.2 APPENDIX B: INJECTION SITE REACTIONS DIARY CARD ................................ ..................... 75 Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 9 LIST OF ABBREVIATION S AND DEFINITIONS OF TERMS Abbreviation or Specialist Term Explanation -HCG -Human chorionic gonadotropin Ad Adenovirus Ad5 Adenovirus serotype -5 Ad5 [E1 -] Ad5 with deletions in the early 1 ( E1) and early 3 ( E3) gene regions Ad5 [E1 -, E2b -] Ad5 with deletions in the early 1 ( E1), early 2b ( E2b), and early 3 (E3) gene regions AE Adverse event AESI Adverse event of special interest ALT Alanine aminotransferase AST Aspartate aminotransferase BSI BioSpecimen Inventory BUN Blood urea nitrogen CAP College of American Pathologists CBC Complete blood count CCR (NCI) Center for Cancer Research CEA Carcinoembryonic Antigen CLIA Clinical Laboratory Improvement Amendments CMI Cell-mediated immunity CMV Cytomegalovirus CR Complete response CRADA Cooperative Research and Development Agreement CRF Case report form CSC Cancer stem cell CT Computed tomography CTCAE Common Terminology Criteria for Adverse Events DFS Disease -free survival DCR Disease control rate DLT Dose -limiting toxicity DLTs Dose -limiting toxicities E1 Adenovirus early 1 gene E2b Adenovirus early 2b gene Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 10 Abbreviation or Specialist Term Explanation E3 Adenovirus early 3 gene ECG Electrocardiogram ECOG Eastern Cooperative Oncology Group EGFR Epidermal growth factor receptor ELISA Enzyme -linked immunosorbent assay EOS End of study ETBX -011 Ad5 [E1 -, E2b -]-CEA ETBX -051 Ad5 [E1 -, E2b -]-Brachyury ETBX -061 Ad5 [E1 -, E2b -]-MUC1 ETBX -081 Combination of ETBX -011 + ETBX -051 + ETBX -061 Vaccines FDA Food and Drug Administration FFPE Formalin -fixed paraffin -embedded GCP Good Clinical Practice GLP Good Laboratory Practice GM-CSF Granulocyte -macrophage colony -stimulating factor cGMP Current Good Manufacturing Practice HBV Hepatitis B virus HCV Hepatitis C virus HHS Health and Human Services HIV Human immunodeficiency virus HPV Human papilloma virus HTD Highest tested dose ICH International Conference on Harmonization IEC Independent Ethics Committee IFN Interferon IHC Immunohistochemistry IL Interleukin INR International normalized ratio IRB Institutional Review Board ISH In situ hybridization LLD Longest lesion diameter LLN Lower limit of normal Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 11 Abbreviation or Specialist Term Explanation MDSC Myeloid -derived suppressor cell MedDRA Medical Dictio nary for Regulatory Activities MRI Magnetic resonance imaging MTD Maximum tolerated dose MUC1 A Transmembrane glycoprotein MUC1c A mod ified MUC1 with agonist epitope NCI National Cancer Institute NIH National Institutes for Health NK Natural kill er (cell) ORR Objective response rate OS Overall survival PBMC Peripheral blood mononuclear cell PD Progressive disease PFS Progression -free survival PR Partial response PT Prothrombin PTT Partial thromboplastin time aPTT Activated partial thromboplastin time RECIST Response Evaluation Criteria in Solid Tumors SAE Serious adverse event SC Subcutaneous sCD27 Soluble CD27 sCD40L Soluble CD40L SD Stable disease SOC System Organ Class SRC Safety Review Committee SUSAR Suspected unexpected serious adverse reactions TAA Tumor -associated antigen Treg Regulatory T cells ULN Upper limit of normal VP Virus particle s Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 12 1 INTRODUCTION 1.1 STUDY OBJECTIVES AND ENDPOINTS 1.1.1 Primary Objective s To determine the overall safety and recommended phase 2 dose of a combination of three immunotherapeutic vaccines (ETBX -011, ETBX -061, and ETBX -051), when administered subcutaneously (SC) to subjects with advanced solid tumors . 1.1.2 Secondary Objectives To make preliminary assessments of : • Objective response rate (ORR) , • Disease control rate (DCR), • Duration of response, • Progression -free su rvival (PFS) • Overall survival (OS) in subjects with advanced solid tumors treated with the combination ETBX -011, ETBX -051, ETBX -061 vaccine regimen . 1.1.3 Exploratory Objectives To evaluate the ability of the combination ETBX -011, ETBX -051, ETBX -061 vaccine regimen to generate T -cell responses specific for Brachyury , MUC1, and CEA . 1.1.4 Primary Endpoints • Dose -limiting toxicities (DLTs) and maximum tolerated dose (MTD) or highest tested dose (HTD). • Treatment -emergent adverse event (AEs) and serious adverse events (SAEs). • Clinically sign ificant changes in safety laboratory tests, physical examinations, electrocardiograms (ECGs), and vital signs. 1.1.5 Secondary Endpoints • Objective response rate (ORR; confirmed complete or partial response) according to the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1. • Disease control rate (DCR; confirmed response or stable disease [SD] lasting for at least 6 months). • Duration of response. • PFS • OS 1.1.6 Exploratory Endpoints • Immunogenicity via the induction of antigen -specific T cells of the combina tion ETBX -011, ETBX -051, ETBX -061 vaccine regimen by flow cytometric analysis, and analysis in sera of sCD27 and sCD40L. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 13 1.2 BACKGROUND AND RATIONAL 1.2.1 Overall Objective As a result of the discovery of new biomarkers associated with tumor development and metastasis, many tumor -associated antigens (TAAs) are being utilized in immunotherapeutic modalities designed to induce anti -tumor directed cytotoxic immune responses. It is increasingly clear that not any one of these TAA is sufficient, as a single entity, by which one can develop a potent immunotherapeutic response. Furthermore, the addition of immune checkpoint inhibitors has augmented the immunotherapy approach against TAAs. Consequen tly, our efforts are focused on developing multi -targeted approaches. The overall goal of the current project is to expand our immunotherapeutic approach for the treatment of advanced cancer employing a multi -targeted approach. Safety and ability to genera te CEA -specific T -cell responses in metastatic colorectal cancer (mCRC) patients using our Ad5 [E1 -, E2b -]-CEA(6D) (ETBX -011) immunotherapeutic as a single agent has been achieved in a clin ical setting (1, 2). Patients in that study exhibited evidence of a favorable survival probability , with all 25 patients treated at least 2 times with vaccine exhibiting a 12 -month ove rall sur vival probability of 48%, with a mean overall survival of 11 months. The phenotypic heterogeneity in terms of expression of different TAAs in a given primary or metastatic tumor mass is a well -established phenomenon (3-7). One can speculate that the use of an immunotherapeutic vaccine regimen targeting three distinct TAAs, each of which is widely expressed on the majority of human carcinomas, woul d be potentially therapeutically advantageous over the use of a vaccine targeting only one TAA. With the safety and immunogenicity of Ad5 [E1 -, E2b -]–CEA established in patients as a single agent, a multi -target approach is now being investigated. The objective is to develop a combination immunotherapeutic approach designed to induce broad anti -tumor immune responses directed against tumors that over express CEA, MUC1 , and/or Brachyury . Preclinical studies were recently published indicating that this multi -targeted CEA, MUC1, Brachyury Ad5 combination vaccine induces immune responses directed against all three target CEA, MUC1, and Brachyury antigens with minimal to no “antigenic competition” in human in vitro studies or in murine vaccination studies (8). Testing of a combination multi -targeted CEA, MUC1, and Brachyury targeted adenoviral vector -based vaccine s in a phase 1 clinical trial is planned to test the safety , ability to generate T -cell responses to each tumor antigen, and effi cacy of the immunotherapeutic. Subsequent trials will involve the use of this vaccine in combination with checkpoint inhibitor monoclonal antibodies (MAbs) and other immune modulators. 1.2.2 Brachyury Expression in Cance r and It s Use as an Immunotherapy Target Brachyury is an embryonic transcription factor of the T -box family that regulates the formation of the posterior mesoderm in the developing embryo, a process that requires the conversion of epithelial cell layers i nto mesenchymal cells (9). While in the majority of adult normal tissues Brachyury is undetectable, with the exception of low levels found in normal testis, thyroid and a subset of B cells (10, 11), aberrantly high levels of Brachyury have been observed in the primary and/or metastatic sites of non -small cell (NSCLC) and small cell (SC LC) lung cancer (12, 13), colon (14), hepatocellular (15), prostate (16) and breast carcinomas (17), including triple negative breast cancer (TNBC) (18). High levels of Brachyury are also characteristic of the rare tumor type chordoma (19, 20), which is thought to originate from remnants of the embryon ic notochord where Brachyury is normally found. Recent studies have now characterized the role of Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 14 Brachyury in the biology of carcinomas and demonstrated the ability of this transcription factor to drive the phenotypic conversion of tumor cells from an epi thelial to a mesenchymal -like phenotype (also designated as an epithelial -mesenchymal transition, EMT, or carcinoma “mesenchymalization”) (21, 22). Carcinoma cells undergoing t his phenotypic transition exhibit enhanced motility and invasiveness in vitro , propensity to metastasize in vivo , and features of tumor stemness (23), including resistance to a range of therapeutics such as chemotherapy, radiation, small molecule therapies and, potentially, immunotherapy (24-27). In agreement with a role for Brachyury in the progression of carcinomas, multiple studies have now shown that the level of Brachyury in the primary tumor correlates with poor patient prognosis in carcinomas of the lung (28), colon (14), breast (17), triple negative breast (18) and gastrointestinal stromal tumor (GIST) (29). Brachyury expression has also been shown to be correlated with advanced stage prostate cancer (16). Transcription factors have been considered “difficult to drug” due to their primary location in the nucleus and lack of a hydrophobic groove for drug attachment. Studies have shown, however, that Brachyury -specific T cells can be generated both in vitro and in vivo (30, 31). Utilizing 9 -mer peptides of the Brachyury protein, for example, Brachyury -specific CD8+ T cells have been expanded in vitro from the blood of cancer patients; these Brachyury -specific CD8+ T cells were utilized in cytotoxic assays for effective lysis of human tumor cells that endogenously express Brachyury (30, 31). The inherent immunogenicity of Brachyury was also revealed from the analysis of immune responses in cancer patients immunized against CEA or PSA. In addition to generating responses against the tumor -associated antigens contained within their respective vaccines, development of Brachyury -reactiv e CD8+ T cells was also observed (32). This expansion of Brachyury -specific T cells may have been the result of cross -presentation of the antigen to the immune system, following tumor destruction in response to the vaccine. These studies demonstrated that Brachyury is immunogenic, and has the potential to function as a target for anticancer vaccination. In addition, two recently completed Phase I clinical studies with a recombinant yeast -Brachyury vaccine (33) or an MVA -Brachyury -TRICOM vaccine (33, 34) also demonstrated the generation of Brachyury - specific T cells as well as safety in humans, thus providing further evidence of immunogenicity (35). These combined properties, i.e., tumor -restricted expression, relevant function in tumor progression, and immunogenicity, make Brachyury a potential target for immunotherapy -mediated approaches against cancer. Preventing or revert ing the EMT process in carcinomas via the use of Brachyury -based cancer vaccines represents an attractive modality to minimize tumor dissemination and the emergence of therapeutic resistance. 1.2.3 CEA Expression in Cancer and Its Use as an Immunotherapy Target CEA represents an attractive target for immunotherapy since it is over expressed on all metastatic colorectal cancer (mCRC) adenocarcinomas and can be overexpressed in other cancers including breast, lung, gastric, pancreatic, bladder, medullary thyroid, h ead and neck, cervical, hepatic, lymphoma, and melanoma. It has also been identified as one of the priority cancer antigens most likely to be successful at generating a cancer immunotherapeutic (36). A reason that CEA is a good target for T cell -mediated immunity in humans is that it contains known epitopes recognized via an MHC -restri cted fashion by human cytolytic T lymphocytes (CTL) that bind to MHC loci HLA - Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 15 A2, A3, and A24 (8, 37). Of the HLA -A2 restricte d epitopes of CEA, CAP1, a nine amino acid sequence, has been reported to stimulate CTL in cancer patients immunized with vaccinia -CEA. CAP1(6D) is a peptide analog of CAP1. Its sequence includes a heteroclitic (non -anchor position) mutation, resulting in an amino acid change from asparagine (Asn) to aspartic acid (Asp), to enhance recognition by the T -cell receptor without any change in binding to HLA A2. Compared with the non -mutated CAP1 epitope, CAP1(6D) has been shown to enhance sensitization of CTL by 100 to 1,000 times (37). The CAP1(6D) epitope has been incorporated into the vector platform (Ad5 [E1 -, E2b -]-CEA(6D)) and has been safely tested in mCRC patients to induce active cell - mediated immunity (CMI) against CEA (1, 2). 1.2.4 MUC1 Expression in Cancer and Its Use as an Immunotherapy T arget MUC1 (CD227) is a TAA that is overexpressed on a majority of human carcinomas and several hematologic malignancies (38-41). MUC1 is normally expressed at the surface of glandular epithelial cells (42) and, in carcinomas, it is overexpressed and aberrantly hypoglycosylated (40, 42, 43). Several clinical trials have been and are being performed to evaluate the use of MUC1 in immunotherapeutic vaccines (2, 44-46). Some of these trials have indicated that targeting MUC1 is safe and may provide survival benefit (2, 45, 47). Multiple enhancer agonist epitopes were previously identified , several of which are in the MUC1 C -terminus region (48, 49). This is potentially important because studies (47) have demonstrated that the C -terminus of MUC1 has oncogenic potential, associates with poor prognosis and drug resistance, and induces “stemness” features in a range of human carcinomas. The human T -cell lines generated using these MUC1 agonist epitopes were more efficient than those generated with the corresponding native epitopes in terms of antigen -specific interferon (IFN) –γ production and lysis of tumor ce lls endogenously expressing native MUC1 (48, 49). Therefore, it is believe d that MUC1 containing modified agonist epitopes has a greate r potential as an immunogenic agent for vaccine development. 1.2.5 Adenovirus -Based Vectors Adenoviruses (Ads) have emerged as leading candidate vectors to deliver vaccines designed to induce CMI and antibody responses (50-52). Ad vectors infect multiple cell types, including dendritic cells which result in priming of a vigorous e response. A ds are a family of DNA viruses characterized by an icos ahedral, non -enveloped capsid containing a linear double -stranded genome (51, 52). None of the human Ads are associated with neoplastic disease and only cause relatively mild, self-limiting illness in immunocompetent individuals. Ad serotype -5 (Ad5) is the most widely used subtype for human vaccines. The wild -type Ad5 genome is approximately 36 kilobases and encodes genes that are divided into early and late viral functions, dep ending on whether they are expressed before or after DNA replication. Ad5 vectors do not integrate (i.e., their genomes remain episomal), so the risk for insertional mutagenesis and/or germ -line transmission is extremely low if at all. 1.2.5.1 Early Generation Ad5 Vectors Early generation Ad5 vectors (Ad5 [E1 -]) contain deletions in the early 1 ( E1) gene and early 3 (E3) gene regions (50-53). The E1 gene is required for DNA synthesis, capsid protein expression, and viral replication, and the E3 gene is required for anti -host immunity. Ad 5 [E1 -] vectors have a decreased ability to replicate and cannot produce infectious virus in cells that do not express the Ad5 E1 genes. Recombinant Ad5 [E1 -] vectors are propagated in human cells (typically human embryonic kidney 293 cells), allowing for Ad5 [E1 -] vector replication and packaging (51). There Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 16 have been over 300 human clinical trials that utilized Ad5 [E1 -] vectors, with more than 2000 subjects given the virus SC, intramuscularly, or intravenously (52). Ad5 [E1 -] vectors have a number of positive attributes; one of the most important is their relative ease for scale up and current good manufacturing practices (cGMP) production. Furthermore, recombinant Ad5 [E1 -] vectors have a large carrying capacity that approaches 7 kilobases. However, preclinical and clinical studies have demonstrated that pre -existing immunity against Ad5 can be an inhibitory factor to the use of Ad5 [E1 -] vaccines in practice (51, 53). Most humans have antibodies against Ad5, with up to two -thirds having lymphoproliferative responses against Ad5 (54, 55). CMI directed against pre -existing or newly synthesized Ad5 cell surface proteins interfere with Ad5 [E1-] vaccines by eliminating the vaccine and vector -infected cells. This reduces the effectiveness of the early generation Ad5 [E1-]-based vaccines. 1.2.5.2 New Generation Ad5 Vectors (Ad5 [E1 -, E2b -]) A new and advanced generation of Ad5 vectors has been developed that, in addition to deletions in the E1 and E3 gene regions, have deletions in the early 2b ( E2b) gene regions (Ad5 [E1 -, E2b- ]) (56-59). The E2b genes are required for viral replication and encode viral DNA polymerase as well as the preterminal protein. In addition, the deletion of the E2b genes drastically reduces late gene expression (capsid type proteins), which decreases anti -vector immune res ponses and enables longer term transgene expression with enhanced immunogenicity. Thus, the Ad5 [E1 -, E2b-] vector overcomes limitations of early generation vectors, as it permits the immunization of people who have been previously exposed to Ad5. In precl inical studies of cancer and infectious disease, Ad5 [E1 -, E2b -] vector -based vaccines were used in multiple homologous immunization regimens and induced immune responses despite the presence of pre -existing Ad5 immunity (2, 8, 59-68). 1.2.6 Clinical Experience with an Ad5 [E1 -, E2b -]-based Cancer Immunotherapy Vaccine 1.2.6.1 Clinical Safety Etubics Corporation has performed a Phase I/II clinical trial (IND#14325) with an Ad5 [E1 -, E2b - ]-based vector containing a modified carcinoembryonic antigen (Ad5 [E1 -, E2b -]-CEA(6D)) for the immunotherapy of CEA expressing cancer (1, 2). The Phase I/II study consisted of a dose - escalation study of four dosage levels (1x109, 1x1010, 1x1011, 5x1011 VP/dose) of ETBX -011 (Phase I component), and the maximally tolerate d dose of ETBX -011 (Phase II and 5x1011 VP/dose components ). Ad5 [E1 -, E2b -]-CEA(6D ) was administered by SC injection every 3 weeks. Thirty -two patients with metastatic colorectal cancer (mCRC) , median age 57.5 (range 38 – 77) who had failed a median of thre e prior chemotherapeutic regimens (range 2 –5), had a performan ce status of 90% (range 70 –100%), and had three sites of metastatic disease (range 1 – 4), were enrolled. The majority of patients were able to receive all three immunizations. Four patients who s topped immunizations early did so due to significant disease progression. A total of 94 immunization treatments was administered to all patients. There was no dose -limiting toxicity and no serious adverse effects (SAE) that resulted in treatment discontinu ation at any vaccine dose level. The most common toxicity was a self -limited, injection site reaction. Other reactions occurred with less than a 10% incidence of all adverse effects (AE) reported and included fever, flu-like symptoms, anorexia, chills, nau sea, and headache. These symptoms were also self -limiting and did not require intervention other than symptomatic measures such as acetaminophen (1, 2). Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 17 1.2.6.2 Immune Response s A secondary objective of the Ad5 [E1 -, E2b -]-CEA(6D ) Phase I trial was to evaluate CEA -specific immune re sponses following vaccination . As determined by an ELISA technique (1, 2), no antibody activity directed against CEA was observed . CEA -specific cell -mediated immunity (CMI ) responses were observed in colorectal cancer patients treated in cohort 1, cohort 2, cohort 3/Phase II, and cohort 5. PBMCs were isolated prior to immunotherapy treatment and after all treatments as wel l as 3 weeks following the la st treatment from patients. CEA -specific ELISpot assays were performed on PBMC as previously described (1, 2, 61) to determine the numbers of interfer on gamma (IFN -) secreting lymphocytes (SFC) after exposure to CEA peptides in vitro . This analysis revealed a dose response to increasing levels of vaccine (Figure 1). The highest CMI levels occurred in patients that received the highest dose of 5x1011 VP (Cohort 5). In(1), a population of polyfunctional CD8+ T cells (those that secrete more th an 1 cytokine when activated) was identified that after immunizations secreted multiple cytokines, a sign of greater functionality of T cells induced by the vaccine. In further follow -up analysis (1) of a few patient blood samples, a decrease in CEA -directed immune responses was noted after immunotherapy immunizations was stopped . This observation supports a rationale for booster immunizations to maintain immune responses. Anti-Ad5 antibo dy (Ab) and CMI against Ad5 were correlated with CEA -specific CMI. Each patient had their serum and PBMC sample tested at baseline (prior to treatment) and at 9 weeks after completion of 3 treatments. Nineteen of 31 colorectal cancer patients (61%) tested in this study had Ad5 neutralizing activity in serum samples prior to the onset of treatment with Ad5 [E1 - , E2b -]-CEA(6D). The mean pre -treatment Ad5 Ab titer value obtained among all patients was 1:189 1:71 SEM and the mean pre -treatment Ad5 Ab titer amo ng seropositive patients was 1:308  1:108. Analysis of serum samples from patients who receive d 3 immunizations revealed Ad5 Ab titers that were significantly increased (P<0.0001, Mann -Whitney test ) by week 9 (mean 1:4767  1:1225 SEM) when compared with their respective baseline values. Analysis of PBMC for CMI responses to Ad5 also revealed a significant increase (P<0.01, Mann -Whitney test) in Ad5 -directed CMI responses after immunizations with Ad5 [E1 -, E2b -]-CEA(6D) (22.6 ± 9.3 SEM IFN -γ secreting SFC at week 0 versus 191.1 ± 83.7 IFN - γ SFC at week 9). Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 18 1.2.6.3 Analysis of Clinical Acti vity The Ad5 [E1 -, E2b -]-CEA(6D ) vaccinated heavily pretreated colorectal cancer patients (total=32) were followed for survival and Kaplan -Meier plots and survival proportions performed (PRISM software) (1, 2). Events were determined by information from the social security death index (SSDI) database, clinical charts and telephone calls ( Figure 2). The seven patients in cohorts 1 and 2 experienced a 12 -month survival proportion of 29%. The 21 patients in cohort 3 and Phase II experienced a 12 -month survival proportion of 48%. The six patients in cohort 5 experienced a 12 -month survival proportion of 50%. Twenty -nine-month overall survival of the intent -to-treat population (32 patients) was 20% ( Figure 2a) with a median survival time of 11 months from informed consent/first injection. For the subset of 28 patients that received all 3 immunizations, the 29-month survival was 23% ( Figure 2b) with a median survival time of 13 months. For the 22 patients optimally dosed with the two highest doses of vacci ne (1 and 5 x 1011) and receiving all 3 immunizations, the 28 -month overall survival was 19% ( Figure 2c). Median overall survival was 13 months in the optimally treated patients. Since there was no active control group in the study, comparisons for significance in survival time cannot be made. There were 3 stable disease events observed immediately after completion of treatment. PrePostPrePostPrePostPrePost0100200300400500 Cohort 1 Cohort 2 Cohort 3/Phase II Cohort 5IFN SFC per 106 PBMC Figure 1: CEA-specific T -cell responses (C MI) in treated patients . CMI (IFN - secretion) was assessed at baseline (Pre) and after a dministrations of Ad5 [E1 -, E2b -]-CEA(6D) (Post). The highest CMI responses (regardless of time point) observed in the patients after treatment revealed a dose response. The highest CMI levels occurred in patients that received the highest dose of 5x1011 VP (Cohort 5). The CMI responses for Cohort 3/Phase II and Cohort 5 were significantly elevated (P=0.0002 and P=0.0317, respectively; Mann -Whitney test ) as compared to their baseline (Pre) values. Specificity of the responses was demonstrated by the lack o f reactivity with the irrelevant antigens - galactosidase and HIV -gag (data not shown). For positive controls, PBMCs were exposed to concanavalin A (data not shown). SFC: spot forming cells. Values = Mean  SEM for each Cohort. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 19 1.2.7 ETBX -081 Pre clinical Studies Recombinant Ad5 [E1 -, E2b -]–CEA , recombinant Ad5 [E1 -, E2b -]–MUC1 and recombinant Ad5 [E1-, E2b-]–Brachyury were generated and characterized as previously described (8). As seen in Figure 3A, Western blot analysis using an anti -Brachyury –specific monoclonal antibody (MAb 54-1)(11) revealed Brachyury expression when human dendritic cells ( DCs) were infected with Ad5 [E1 -, E2b -]–Brachyury . An Ad5 [E1 -, E2b -] vector devoid of any transgene (Ad5 [E1 -, E2b - ]–null) was used as a negative control and SW620 human colon carcinoma cells that endogenously express Brachyury were used as a positive con trol. An anti -MUC1 –specific MAb was used to detect the expression of MUC1 in Ad5 [E1 -, E2b -]–MUC1 –infected human DCs ( Figure 3B). SW620 cells, which also express MUC1 endogenously, were used as a positive control. The difference in molecular weights seen in the human DCs versus the SW620 human carcinoma cells is most likely due to the differential glycosylation of the MUC1 protein. As has been previously shown by others (69-72), it would appear that MUC1 -C is being expressed in the human DCs predominantly as the unglycosylated 17 or 15 kDa form and not the 25 -20 glycosylated species. A Western blot of Ad5 [E1 -, E2b -]–CEA infected human cell s is shown in Figure 3. The generation of Brachyury -, CEA -, and MUC1 -specific human CD8+ T cells employing the corresponding peptide for each TAA was previously reported (30, 31, 37, 48, 49, 73). As shown in Table 1, Ad5 [E1 -, E2b -]–null did not activate any of the T cells to produce IFN -γ. Ad5 [E1 -, E2b - ]–Brachyury –infected DCs activated Brachyury -specific T cells and not CEA -specific T ce lls (as a negative control). This demonstrates that the Ad5 [E1 -, E2b -]–Brachyury –infected DCs could process Brachyury in a manner that generates Brachyury –MHC Class I complexes capable of specific T -cell activation. Similarly, Ad5 [E1 -, E2b -]–CEA –infected DCs specifically activated CEA -specific T cells but not MUC1 -specific T -cell lines. Both Class I HLA -A2 and -A24 MUC1 - specific T -cell lines have been previously generated (49) and the Ad5 [E1 -, E2b -]–MUC1 –infected 0 4 8 12 16 20 24 28020406080100a MonthsPercent Survival Proportion 0 4 8 12 16 20 24 28020406080100b MonthsPercent Survival Proportion 0 4 8 12 16 20 24 28020406080100c MonthsPercent Survival Proportion Figure 2: Kaplan -Meier survival plots on long -term overall survival of treated mCRC patients. Panel a represents all treated patients.