1.1.2.2 To evaluate evidence of clinical benefit, such as progression- free survival, RECIST criteria, reduction in serum markers, and/or reduction in circulating tumor cells. 1.1.2.3 Parameters of general immune activation: frequency of immune cell subsets in peripheral blood and changes in serum levels of cytokines and antibodies to brachyury . 1.1.2.4 Correlation of brachyury expression in tissue samples with clinical outcomes. 1.2 BACKGROUND AND RATIONALE MVA-brachyury-TRICOM is a novel recombinant vector- based therapeutic cancer vaccine developed by the LTIB in collaboration with our CRADA partner Bavar ian Nordic, Inc. The vaccine is designed to enhance the immune response against brachyury, which is overexpressed in many solid tumor types, including lung, breast, ovarian, prostate, chordoma, colorectal, and pancreatic adenocarcinoma, as well as some hematologic malignancies . 1.2.1 Description and Identification of Brachyury Using a computer-based differential display analysis tool to conduct global comparison of expressed sequence tag (EST) clusters in the Unigene database, 1,2 the gene encoding for the transcription factor brachyury was identified as highly represented in tumor-derived libraries and rarely observe d in normal tissue- derived libraries.3 Brachyury is a member of the T -box family of transcription factors, characterized by a highly conserved DNA-binding domain des ignated as T-domain.4-7 Brachyury homologs have been reported to be involved in e mbryonic mesodermal development.4,8-11 1.2.2 Brachyury Function in Epithelial- to-Mesenchymal Transition (EMT) Epithelial- to-mesenchymal transition (EMT) is a reversible process during which cells sw itch from a polarized, epithelial phenotype into a highly motile, mesenchymal phenotype.12,13 At the biochemical level, the EMT program involves the downregulation of epithelial proteins such as E-cadherin and cytokeratins and the induction of mesenchymal proteins, including fibronectin, N-cadherin and vimentin. 14,15 Numerous observations support the concept that the EMT process plays a role in the progression of human carcinomas (Fig ure 1).16 11 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential Figure1 . Role of brachyury in epithelial -to-mesenchymal transition during tumor progression We have demonstrated17 that overexpression of brachyury in human carcinoma cell lines is able to drive a switch from an epithelial to a mesenchymal -like phenotype (i.e., EMT). For example, stable transfection of a pancreatic cancer cell line, PANC -1, with a plasmid encoding for full- length human brachyury resulted in cell spreading, decreased epithelial E -cadherin, and increased mesenchymal fibronectin expression (Figure 2). Western blot also confirmed the overexpression of other mesenchymal markers and downre gulation of epithelial markers (Fig ure 2), providing evidence that brachyury may facilitate EMT.17 Over expression of brachyury in epithelial tumor cells also resulted in a concomitant increase in tumor cell migration and extracellular matrix invasion (Fig ure 3A and B). Stable silencing of brachyury expression in brachyury -positive human carcinoma cells (lung H460 cells) resulted in downregulation of mesenchymal markers and upregulation of epithelial markers, with concomitant loss of cell migra tion and invasion (Fig ure 3C and D).17 Figure 2. Brachyury alters morphology and EMT marker expression in human pancreatic cancer epit helial tumor cells. (A) Bright field images of PANC -1-pcDNA (control) and PANC-1- p/brachyury cells and immunofluorescent analysis of EMT markers; merged images with DAPI stained nuclei are shown. (B) Western blot analysis of brachyury - induced EMT markers. See ( 17) for details. 12 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential Brachyury expression also negatively modulated tumor cell- cycle progression and correlated with low expression of cyclin D1, and carcinoma cells silenced for brachyury expression grew at a greater rate in vitro (Figure 4A). These brachyury -silenced cells grew at a comparable rate to control cells when implanted subcutaneously in athymic mice (Fig ure 4B); however, they had a diminished capacity to metastasize to the lungs from the primary, subcutaneous tumor, or to form experimental lung metastases when injected i.v . (Figure 4C). Collect ively, these results demonstrate that the transcription factor brachyury confers on the tumor cells a mesenchymal phenotype as well as migratory and invasive cap abilities and enhances tumor cell progression.17 1.2.3 Analysis of Brachyury Expression in Human Tumors and Normal Tissues By using reverse transcription followed by polymerase chain reaction (RT- PCR), investigators in the LTIB have identified the overexpression of brachyury in gastrointestinal, Figure 3. Brachyury induction of migration and invasion. (A and B) Brachyury induces a highly migratory and invasive EMT phenotype in vitro in human PANC -1 pancreatic carcinoma cells. (C and D) Silencing brachyury expression in H460 human lung carcinoma cells reduces both migratory and invasive capacity. See ( 17) for details. Figure 4. Brachyury inhibition with silencing RNA reduces tumor metastases. Compared to control (con.shRNA, circles), human H460 lung carcinoma cells silenced for brachyury (Br.shRNA, triangles) (A) proliferate at a faster rate in vitro (p = 0.003); (B) grow at a similar rate when injected s.c. in athymic mice; and (C) form less experimental metastases when injected intravenously (p = 0.002).