Identification of tumor associated immune responses against brachyury, a transcription factor and driver of EMT, in chordoma patients receiving a yeast-brachyury vaccine (gi-6301)

Methods An expansion cohort of 7 patients with chordoma, enrolled in the Phase I clinical trial “Open Label Study to Evaluate the Safety and Tolerability of GI-6301 (Whole Heat-Killed Recombinant yeast Modified to Express Brachyury Protein) in Adults with Solid Tumors”, NCT01519817, were assessed for brachyury-specific T cell responses. Patients received 40 yeast units of vaccine every 2 weeks, and monthly dosing following restaging at day 85. PBMCs from preand post-vaccination were cultured in a 7-day in vitro stimulation (IVS) with overlapping 15-mer peptides of the brachyury protein that was encoded in the vaccine, and IL7/IL15. Following the IVS, cells were rested for 4 days, and then re-stimulated with 15-mer peptides. HLA 15-mers and a mixture of 9-mer to 15-mers of CMV, EBV, Flu, and Tetanus Toxin (CEFT) served as negative and positive controls, respectively. Brachyury-specific T cell responses were analyzed by flowcytometry intracellular staining (ICS) of CD4 and CD8 T lymphocytes for the cytokines IFN-g, TNF, and IL-2, and the perforin/granzyme marker CD107a. Cells positive for ≥2 cytokines were considered multipotent T lymphocytes, while cells co-expressing at least one cytokine and positive for CD107a were classified as having a cytokine/lysis association. A patient was considered an immune responder if, after brachyury 15-mer IVS, the frequency of T lymphocytes positive for a cytokine or CD107a postvaccine was >50% compared to both (1) pre-vaccine values and (2) HLA control post-vaccine.