No patients discontinued treatment due to a drug- related adverse event. In the BNIT -BR-003 study, one event of c ellulitis in one patient was the only SAE reported and was considered possibly related. In the BNIT- BR-003 study, immunotherapy-induced HER-2 transgene- specific and MVA vector -specific T -cell and humoral responses were detected during and after treatment, indicating that MVA -BN-HER2 is immunologically active in the ad juvant setting of breast cancer. MVA- BN-PRO was evaluated in prostate cancer patients enrolled in a single phase I clinical trial, BNIT -PR-001. The primary objective of this study was to evaluate the safety and tolerability of single and multiple injection regimens of MVA -BN-PRO for the treatment of androgen -insensi tive prostate cancer. There were no dose- limiting toxicities identified and no drug- related serious adverse events noted in 24 patients treated. 1.2.15 TRICOM Costimulatory Molecules Destruction of immunological targets requires T -cell lymphocyte recognition, via the T- cell receptor, of antigenic peptides presented in the context of MHC mol ecules on APCs. In addition to this antigen -specific signal , a second, antigen-independent signal is required for T- cell activation . 32,33 This second signal is provided by the interaction of specific ligands on the T -cell surface with costimulatory molecules expressed on APCs. The most extensively studied pathway of costimulation is that involving the interaction of the costimulatory molecule B7.1 expressed on APCs with CD28 and CTLA4 on the T cell.34,35 A number of additional costimulatory molecules on APCs have been identified, including ICAM -1 and LFA -3, whos e ligands are LFA -1 and CD2, respectively, on the surface of T cells.36,37, 38 Proper engagement of the T -cell receptor and costimulatory receptor s requires the expression of both antigen and costimulatory molecules in the same cell. Therefore, coexpression of costimulatory molecules using a single recombinant vector presents the potential of cooperation among these proteins to enhance T-cell activation. Recombinant vectors coexpressing 3 costimulatory molecules, LFA -3, ICAM -1, and B7.1, designated TRICOM™, have been shown to have synergistic effects on antitumor responses compared to vectors expressing individual costimulatory molecules. 33 Mice immunized with a recombinant vaccinia virus coexpressing carcinoembryonic antigen (CEA) and murine TRICOM exhibited greater immune responses and antitumor responses than mice immunized with a recombinant vaccinia virus coexpressing CEA and murine B7.1. Enhanced antitumor immunity was also observed in mice that were transgenic (tolerant) for CEA . 39 MVA-brachyury- TRICOM , therefore, has been designed to simultaneously express brachyury together with B7.1, LFA- 3, and ICAM -1. 1.2.16 MVA- TRICOM in CLL We previously demonstrated that infection of CLL cells with MVA expressing the costimulatory molecules B7 .1, ICAM -1, and LFA-3 (TRICOM) increased expression of these costimulatory 24 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential molecules on the surface of CLL cells and thus augmented their antigen -presenting capability. We further evaluated an alternative MVA vector platform encoding for human CD40L in comparison with MVA-TRICOM for their ability to enhance the immunogenicity of CLL cells, in vitro . Our results indicated that MVA- TRICOM -infected and MVA -CD40L -infected CLL cells are equ ally potent at inducing autologous T- cell proliferation. 40 1.2.17 Infection of Human Dendritic Cells with MVA -Brachyury -TRICOM In order to investigate the ability of MVA -brachyury -TRICOM to infect cells from the blood of normal donors, dendritic cells (DCs) were prepared from peripheral blood mononuclear cells (PBMCs) by culture for 6 days in the presence of GM -CSF and IL-4, and subsequently incubated with wild -type MV A (MVA- WT), MVA -TRICOM (expressing only the 3 costimulatory molecules), or MVA -brachyury -TRICOM. Using MVA -WT alone, no increased expression of CD80, CD54, or CD58 was observed. MVA- TRICOM resulted in upregulation of each of the 3 costimulatory molecules . Addition of the target antigen, brachyury , did not interfere with upregulation of the costimulatory molecules, indicating that all 4 transgenes could be inserted into the vector and result in effective infection and gene expression (Table 4). Table 4. In vitro infection of human DCs with MVA-brachyury- TRICOM Brachury expression was present w hen DCs were infected with MVA -brachyury -TRICOM , but was undetectable in uninfected or MVA- WT-infected DCs (Figure 9). Similarly, expression of brachyury in DC s infected with MVA -brachyury-TRICOM (MOI = 10) was evaluted by immunofluorescence using a monoclonal anti-brachyury Ab (Abcam) (Figure 10). 25 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential These results indicate that the MVA -brachyury-TRICOM vector is able to infect human DC s, resulting in upregulation of brachyury as well as 3 costimulatory molecules (TRICOM) . 1.2.18 Induction of Human T C ells by MVA -Brachyury -TRICOM In order to investigate the ability of MVA -brachyury -TRICOM to expand brachyury -specific T cells from the blood of normal donors, DCs were prepared from PBMCs and infected with MVA- WT, MVA- TRICOM, or MVA-brachyury -TRICOM vectors. Brachyury- specific T cells were stimulated with irradiated DCs. Supernatants were collected and evaluated for IFN -gamma production by ELISA assay. Shown is the IFN-gamma production in response to MVA- brachyury -TRICOM vs . MVA- WT-infected DCs (Figure 11). Figure 9. Detection of brachyury expression by Western blot analysis. Expression of brachyury was evaluated by Western blot analysis with a monoclonal rabbit anti -brachyury Ab (clone 54-1). GADPH expression is also shown.