Figure 10. Detection of brachyury by immunofluorescence. Expression of b rachyury was evaluated by immunofluorescence with a monoclonal anti -brachyury Ab (Abcam). ( Green indicates b rachyury expression, gray indicates DAPI -stained nuclei. ) 26 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential Figure 11. Brachyury -specific CD8+ T cell stimulation by MVA -brachyury -TRICOM -infected DCs compared with MVA- TRICOM -infected DCs. 1.2.19 MVA- Brachyury -TRICOM -Infected Hum an DCs Stimulate Brachyury -Specific CD 4+ T cells CD4+ T cells from normal -donor PBMCs were exposed to either MVA- WT or MVA -brachyury - TRICOM -infected DCs and then exposed to a control (HSA) or brachyury protein. The CD4+ T cells exposed to MVA -WT did not proliferate in response to HSA or brachyury, but t he CD4+ T cells exposed to MVA -brachyury -TRICOM DCs proliferated significantly better when exposed to brachyury protein compared with HSA (Figure 12). These results indicate MVA -brachyury - TRICOM is able to e ffectively expand CD4+ brachyury-specific T cells from the blood of normal donors, as compared to the MVA-WT vector. 27 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential Figure 12. MVA -brachury -TRICOM (and not MVA- WT) -infected human DCs expand CD4+ T cells from PBMCs of normal donors that recognize purif ied brachyury protein . 1.2.20 MVA-B avarian Nordic (MVA -BN) MVA- BN effectively infects mammalian cells. Infection of mammalian cells results in transcription of the viral genes, but no MVA- BN is released from the cells due to a genetic block in the viral assembly and egress. The infected cells eventually undergo apoptosis (programmed cell death). MVA -BN replicates efficiently in CEF cells and probably also in certain other avian cell lines. Despite its high attenuation and reduced virulence, in preclinical studies MVA- BN has been shown to elicit both humoral and cellular immune responses to vaccinia and genes cloned into the MVA genome. MVA-BN is a potent inducer of type I interferon (IFN) in human cells. Like other MVA, MVA- BN expresses a soluble interleukin -1 rec eptor, 41 which has been implicated as an antivirulence factor for certain poxviruses .42 MVA does not express soluble receptors for IFN -γ, IFN -α/β, tumor necrosis factor , or CC chemokines.43 MVA- BN i s a further attenuated MVA strain that has lost its ability to replicate in most mammalian cell types, including almost all human cell lines , and is safe in severely immun ocompromised animals (AGR129 mice). The hallmark of MVA- BN is the fact that it does not productively replicate in the human keratinocyte cell line HaCat, the human cervix adenocarcinoma cell line HeLa, the human embryo kidney cell line 293 (HEK293), and the human bone osteosarcoma cell line 143B. However, like other MVA strains, MVA- BN effectively infects mammalian cells. 2 ELIGIBILITY ASSESSMENT AND ENROLLMENT 2.1 E LIGIBILITY CRITERIA 2.1.1 Inclusion Criteria (All Subjects) 2.1.1.1 Patients must have a metastatic or unresectable locally advanced malignant solid tumor , histologically confirmed by th e Laborato ry of Pathology, NCI. In the case of chordoma, unresectable, locally recurrent, or metastatic tumors are acceptable for enrollment, given that this represents incurable disease. Efforts will be made, as much as possible, to enroll patients with tumor types with known increased expression of brachyury (such as lung, breast, ovarian, prostate, colorectal, pancreatic, or chordoma; other tumors may be included as data on the level of brachyury in those tumors becomes available). 2.1.1.2 Patients may have measurable or nonmeasurable but evaluable disease.