Figure 3. Epitope modified minigene design. (A–F) To determine the optimum epitope modified minigene (EMM) scaffold, candidate DNA vectors were constructed for the expression of immunogenic carcinoembryonic antigen (CEA) polyepitopes. The helper epitope from Tetanus toxin (p30) is encoded on the majority of the constructs as indicated. (A–B) Ubiquitin (ubi)-fused proteasome-dependent EMM variations in which either AAY (A), or LRA, or RLRA (B) were utilized as a processing spacer between epitopes. (C–F) Furin-dependent EMM variations in which REKR was the selected sequence for furin-specific cleavage and epitope processing. The leader peptide from the secretory protein tissue plasminogen activator (TPA) was used for guiding the polyepitope into the endoplasmic reticulum. (C) Minimal furin-dependent EMM with the p30 helper epitope. (D) The membrane-translocating sequence (MTS) from the HIV-1 derived Tat gene at the C-terminus and the p30 helper epitope. (E) Fusion of the p30 helper epitope and heat-labile toxin B (LTB) subunit of E. coli to the polyepitope C-terminus. (F) LTB fusion only.