Identification and characterization of a cytotoxic T-lymphocyte agonist epitope of brachyury, a transcription factor involved in epithelial to mesenchymal transition and metastasis

Abstract

The transcription factor brachyury is a major driver of epithelial to mesenchymal transition (EMT) in human carcinoma cells. It is overexpressed in several human tumor types vs. normal adult tissues, except for testes and thyroid. Overexpression is associated with drug resistance and poor prognosis. Previous studies identified a brachyury HLA-A2 cytotoxic T-lymphocyte (CTL) epitope. The studies reported here describe an enhancer epitope of brachyury. Compared to the native epitope, the agonist epitope: (a) has enhanced binding to MHC class I, (b) increased the IFN-γ production from brachyury-specific T cells, (c) generated brachyury-specific T cells with greater levels of perforin and increased proliferation, (d) generated T cells more proficient at lysing human carcinoma cells endogenously expressing the native epitope, and (e) achieved greater brachyury-specific T-cell responses in vivo in HLA-A2 transgenic mice. These studies also report the generation of a heat-killed recombinant Saccharomyces cerevisiae (yeast) vector expressing the full-length brachyury gene encoding the agonist epitope. Compared to yeast-brachyury(native) devoid of the agonist epitope, the yeast-brachyury(agonist) enhanced the activation of brachyury-specific T cells, which efficiently lysed human carcinoma cells. In addition to providing the rationale for the recombinant yeast-brachyury(agonist) as a potential vaccine in cancer therapy, these studies also provide the rationale for the use of the agonist in (a) dendritic cell (DC) vaccines, (b) adjuvant or liposomal vaccines, (c) recombinant viral and/or bacterial vaccines, (d) protein/polypeptide vaccines, (e) activation of T cells ex vivo in adoptive therapy protocols, and (e) generation of genetically engineered targeted T cells.

Phase I Study of a Poxviral TRICOM-Based Vaccine Directed Against the Transcription Factor Brachyury

“…Utilizing 9-mer peptides of the brachyury protein, for example, brachyury-specific CD8 + T cells have been expanded in vitro from the blood of cancer patients. These brachyury-specific CD8 + T cells were utilized in cytotoxic assays for effective lysis of human tumor cells that endogenously express brachyury (23,24). These combined properties, i.e., tumor-restricted expression, relevant function in tumor progression, and immunogenicity, make the self-antigen brachyury a potential target for immunotherapy-mediated approaches against cancer.…”

Neutralization of IL-8 decreases tumor PMN-MDSCs and reduces mesenchymalization of claudin-low triple-negative breast cancer

“…NK cells were isolated from healthy donor peripheral blood mononuclear cells by using a magnetic NK Cell Isolation Kit (Miltenyi Biotech) and were cultured in RPMI media containing 10% FBS. Antigen-specific, HLA-A 02 -and HLA-A 24 -restricted cytotoxic T lymphocytes directed against an epitope of brachyury (WLLPGTSTV) or mucin-1 (MUC-1, KYHPMSEYAL), respectively, were generated and expanded as previously described from the blood of cancer patients (80)(81)(82).…”

Aberrant expression of the embryonic transcription factor brachyury in human tumors detected with a novel rabbit monoclonal antibody

“…It has been previously shown that brachyury is immunogenic in humans [12, 35, 36] and based on those studies, a heat-killed recombinant Saccharomyces cerevisiae (yeast) brachyury vaccine and a MVA-poxviral vaccine encoding brachyury and a triad of costimulatory molecules (TRICOM) have been developed and characterized [26] and entered Phase I clinical testing in patients with advanced carcinomas or chordomas [28, 29, 37, 38]. In the context of clinical studies of brachyury-based vaccines, we believe MAb 54-1 could be of potential use to determine what type of tumors express brachyury and could therefore be targets, and to evaluate the presence of brachyury-positive tumor cells pre vs. post-treatment, therefore assisting in the interpretation of the efficacy of brachyury-based therapies.…”