Brachyury expression and prognosis. A B Figure 8. Expression of b rachyury in human carcinoma cells induces resistance to chemotherapy. (A) Silencing of brachyury (sh/b rachyury) in H226 human lung carcinoma cells resulted in enhanced sensitivity to chemotherapy. (B) A549 lung cancer cells stably transfected with a brachyury vector (ph/b rachyury) had enhanced resistance to several chemo therapeutic agents. 19 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential 1.2.8 Immunogenicity of the Brachyury Protein By using an MHC -peptide binding prediction algorithm, a 9- mer HLA -A2 binding epitope of the brachyury protein has been identified,3 which was successfully used in vitro to expand human brachyury -specific CD8 + T-cell lines from peripheral blood of both normal donors and cancer patients. Functional assays demonstrated that these brachyury -specific T -cell lines are able to efficiently lyse, in an MHC -restricted manner, human carcinoma cell lines (breast, lung, colon) endogenously expressing the brachyury protein. Utilizing the identified HLA -A2-binding epitope of brachyury , CD8+ brachyury -specific T -cell responses were detected in the blood of cancer patients following vaccinations with either CEA - or prostate- specific antigen (PSA)-based vaccines , likely due to the phenomenon of antigen cascade.21 All together , these results demonstrated that brachyury is an immunogenic protein in humans. 1.2.9 Prior Human Experience with Brachyury Vaccine Therapy A phase I study using escalating doses of y east-brachyury vaccine (GI-6301) is ongoing at the NCI in 23 subjects with varied malignancies, including chordoma and colorectal cancer. The vaccine has been very well tolerated with no observed DLT s. 1.2.10 Poxviral Vectors Vaccinia virus has been used for over 200 years as a vac cine for smallpox and has a well - established safety profile. The virus actively replicates in human cells, resulting in the presentation of high levels of antigen to the immune system over a period of 1–2 weeks, substantially increasing the potential for immune stimulation. The immune response specific to vaccinia then eliminates the virus. As a result of its safety profile and ability to elicit both humoral and cell-mediated immunity in humans, the vaccinia virus was chosen as one of the vectors to deliver MUC -1, CEA, and TRICOM in previous NCI-sponsored trials. 22,23 Immunization with live recombinant pox viral vectors that have been genetically engineered to express one or more antigens allows expression of tumor- associated antigens (TAAs ) and subsequent co-presentation of antigenic peptides with host histocompatibility antigens, a strategy that favors the induction of cell-mediated immune responses. Recombinant poxviruses can infect antigen -presenting cells, including dendritic cells and macrophages, resulting in efficient expression of TAAs simul taneously with costimulatory molecules required to elicit T- cell responses. TAAs expressed by recombinant poxviruses are presented to the immune system together with highly immunogenic vir al proteins, which may act as adjuvants to enhance immune responses to the TAAs. Thus, the use of recombinant pox viral vectors to present TAAs to the immune system results in the generation of killer T cells that specifically destroy the selected tumor with little incremental toxicity. 20 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential 1.2.11 Safety of Poxviral Vectors A recent r eport on safety data from NCI clinical trials with poxviral vaccines evaluated all vaccine i njections from 215 patients in 8 clinical trials involving poxviral vector vaccines. Vaccines consisted of recombinant vaccinia and recombinant fowlpox encoded with 3 human costimulatory molecules (TRICOM), and prostate specific antigen (PSA), or carcinoembryonic antigen, and/or mucin- 1. Vaccines were administered at doses between 1.2 x 10 8 to 2 x 109 pfu, subcutaneously, in all patients. 84 patients also received ot her concurrent treatment modalities, such as radiation, celecoxib, ipilimumab, samarium-153, or flutamide on 4 of these trials. All 8 clinical trials involved granulocyte-macrophage colony- stimulating factor (GM -CSF) 100 mcg, or recombinant fowlpox encodin g GM -CSF at 1 x 10 8 pfu as an immune adjuvant. G rade 2 or higher adverse events at least possibl y attributed to vaccine were reported (Table 3).