Efficacy of Intracellular Immune Checkpoint-SilencedDendritic Cell Vaccine

In order to construct a recombinant replication-deficient adenoviral vector, designated as AdsiSSF, which coexpresses a small hairpin human SOCS1-siRNA (siSOCS1), dominant negative (DN) survivin and MUC1 fusion protein, and a secretory flagellin, the TLR5 ligand (SupplementaryFig. 1A), we first modified survivin, MUC1, and flagellin genes, as well as identified siRNA that can effectively silence human SOCS1 (5’-CACGCACTTCCGCACATTC3’). Because of the anti-apoptotic biological activity of survivin for the potential promotion of malignant cell survival and transformation, two mutations were introduced into the survivin gene to generate a DN survivin mutant. A substitution mutation of Thr with Ala in position 34 was made to eliminate the p34-cyclin B1 phosphorylation site and anti-apoptotic activity, as described previously(1). Moreover, to avoid possible reverse mutation of the survivin mutant back to a functional phosphorylation site, a 16 amino acid truncation at the N-terminus of survivin was further made since the N-terminal sequence is required for its inhibition of apoptosis (2). We used a short human mucin 1 (MUC1) fragment containing only a three 20 amino acid tandem repeat sequence, because a known dominant T cell epitope is located within the 20 amino acid repeat(3)and full-length or lengthyMUC1 protein was found to have immune suppressive activity by inhibiting a Th1-type CTL response(4). To generate a DN survivinMUC1 fusion gene (DN SM) as target tumor-associated antigens for the Ad-siSSF vector, the double mutated DN survivin was genetically linked in-frame to the three 20 amino acid repeat fragment of MUC1. For facilitating the secretion and subsequent interaction with surface TLR5 on DCs of flagellin by transfected DCs, a signal leader sequence derived from human tyrosinase was genetically linked to the N-terminus of the flagellin gene. We also identified siRNA with the ability to specifically downregulate human SOCS1 (SupplementaryFig. 1B).