See ( 17) for detail s. 13 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential bladder, kidney, ovary, uterus, and testicular carcinomas. Similar studies also found overexpression of brachyury mRNA in cell lines of lung, colon and prostate cancers, but not in the majority of normal tissues te sted, with the exception of expression in the testis and low levels of expression in B cells pooled from multiple normal donors. The expression of brachyury mRNA in normal B cells was further evaluated in CD19+ B- cell fractions isolated from various healthy donors; weak amplification was observed in 4/9 samples analyzed by RT -PCR. These results at the RNA level, however, contrasted with data obtained by immunohistochemistry analysis in normal spleens and lymph nodes, which were negative for the expression of brachyury protein. Moreover, the cytotoxic lysis of normal B cells was evaluated by using brachyury -specific T cells as effectors; no lysis was observed with any of the normal B cells purified from the blood of 5 different healthy donors. 3 We have determined that EBV infection of human B cells enhances brachyury expression; approximately 1 in 105–106 human B cells have latent EBV infection. Immunohistochemistry (IHC ) analysis of brachyury expression using an anti- brachyury monoclonal Ab confirmed the selective expression in tumors of this transcription factor. No expression of brachyury was observed in the majority of normal tissues tested, with the exception of test is (3/3 positive) and thyroid (4/6 positive) . The expression of brachyury protein was also detected in 3 of 4 thyroid tissue lysates evaluated by Western blot. These results at the protein level contrasted with the expression of brachyury at the mRNA level , which was negative in 3/3 individual thyroid tissues tested by RT- PCR. Altogether, these results indicate brachyury expression in 7/13 thyroid tissues analyzed. Immunohistochemistry analysis of normal tissues obtained from non- cancer subjects demonstrated brachyury expression in 0/5 lung, 0/3 heart, 0/3 brain, 0/3 liver, 0/3 kidney, 0/3 spleen, 0/3 skeletal muscle, 0/1 adrenal gland, 0/1 skin, 4/6 thyroid, and 3/3 testis samples analyzed. Quantification of brachyury expression has been performed in multi ple carcinoma tissues by real-time PCR analysis of brachyury mRNA levels. These studies have been performed, for example, with lung 17,18 and breast carcinomas .19 In the same studies and in Hamilton et al,20 PCR analysis of brachyury mRNA expression in multiple normal tissues has also been performed. Unlike wi th carcinomas, the expression of brachyury mRNA was undetectable or very low in the majority of normal tissues evaluated. The only normal tissue that showed brachyury mRNA levels comparable to those found in some carcinoma tissues was the normal testis. An alysis of brachyury protein expression was also conducted by immunohistochemistry.18,19 Expression of brachyury protein was demonstrated in lung carcinomas and invasive ductal breast carcinomas, both primary and metastatic lesions. Unlike with carcinomas, expression of brachyury protein was negative among the normal tissues analyzed, with the exception of test is and some cases of normal thyroid. The transcription factor brachyury is a driver of EMT . This phenomenon, which is responsible for the acquisition of tumor invasiveness and tumor resistance to multiple therapeutics, is a manifestation of tumor plasticit y and therefore is expected to take place in a transient fashion along tumor progression. A few t umor cells in a primary tumor mass might express brachyury at a certain time under the influence of environmental factors such as TGF -β, IL-8, and others, and then disseminate from the primary tumor into circulation and secondary metastatic sites. The 14 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential analysis of a primary tumor biopsy or surgical sample might, therefore, be negative for brachyury expression due to the transient nature of the phenomenon, while disseminated tumor cells might still be positive for expression of the target brachyury . Thus, when analyzing a primary tumor by IHC or PCR, it is difficult to determine whether results reflect the state of individual tumor cells at one point in time, or whether a subpopulation of cells had previously undergone the EMT process via brachyury expression and had already metastasized. Additionally, our IHC studies with matched primary/metastatic tumors demonstrated that primary tumors with focal brachyury expres sion corresponded to metastatic sites highly positive for the expression of brachyury. These observations constitute the rationale for not including the level of expression of brachyury in primary tissue samples as a criterion for the inclusion of patients in the study. 1.2.4 Brachyury Expression in Lung Carcinomas Expression of brachyury was found in approximately 40% of primary lung tumors (Table 1, Figure 5A-D ). Real-time PCR analysis of brachyury mRNA expression in multiple human lung tumor tissues showed that the percentage of tumors positive for brachyury expression increases with tumor stage : 30/48 (62.5%) of s tage II –IV lung cancer biopsies showed overexpression of brachyury vs. 12/32 (37.5%) of stage I lung cancers and 2/16 (12.5%) of histologically nor mal lung biopsies f rom lung cancer patients (Fig ure 6).17 Table 1. Brachyury protein expression analyzed by IHC Lung tumor tissues Brachyury -positive Adeno carcinoma 10/21 (48%) Squamous carcinoma 3/12 (25%) Undifferentiated carcinoma 2/4 (50%) Bronchioalveolar carcinoma Small cell lung cancer 1/1 (100%) 0/1 (0%) Total 16/39 (41%) 15 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential Figure 6A. Brachyury expression in human lung tumor tissues. Real-time PCR was performed for brachyury on lung tumor tissue cDNA from 80 lung cancer patients of t he indicated stages of disease. The stage II, III, and IV cDNA samples are further represented by the symbols , ▲, and , respectively. As controls, 16 samples of “normal” lung cDNA were analyzed, each obtained from a histologically normal section of lung from a lung cancer patient. See (17) for details.