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Immunotherapy, in particular Immune Checkpoint Blockade (ICB) has been regarded as the next standard of care for many solid tumor indications. Yet, a significant proportion of patients do not respond to therapy. More specifically, failure of ICB has been linked to the absence of an anti-tumor immune response that can be boosted or revitalized. Cancer vaccines that are designed to elicit such response by educating the host immune system to recognize tumor antigens, are therefore considered a key next generation therapeutic modality for the treatment of human cancers. Various vaccine platforms have been developed over the years, but recent advances in delivery technologies combined with the intrinsic qualities of the DNA matrix have positioned DNA vaccines as a safe and flexible alternative to other types of vaccine technologies. Vaccibody is developing DNA vaccines that allows specific targeting of tumors antigens to Antigen Presenting Cells (APCs), thus maximizing the elicited immune response. Here we present a second-generation version of our DNA platform in which our vaccibody molecule can be co-expressed with immune-stimulatory proteins from one plasmid using a multicistronic design. Compared to the vaccibody molecule alone the simultaneous expression of selected immune stimulatory cytokines was shown to boost the overall immune response almost 3-fold to drive a potent anti-tumor response. These data demonstrate the flexibility and potential of DNA vaccines as well as the advantages of combining an APC targeted delivery of tumor specific antigens together with a local production of immune stimulatory proteins. Citation Format: Audun Beraas, Pierre Dillard, Judith Jing Wong, Christian Winther Wold, Eirik Solbakken, Linn Guro Oslen, Joel Benjamin Heim, Stalin Chellappa, Agnete Fredriksen, Mikkel Wandahl Pedersen, Stine Granum. A novel and versatile cytokine empowered DNA vaccine platform with superior immune activating potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3558.

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.