Söhnke Voss

A novel RNA-based adjuvant combines strong immunostimulatory capacities with a favorable safety profile

Protein‐ and peptide‐based tumor vaccines depend on strong adjuvants to induce potent immune responses. Here, we demonstrated that a recently developed novel adjuvant based on a non‐coding, long‐chain RNA molecule, termed RNAdjuvant®, profoundly increased immunogenicity of both antigen formats. RNAdjuvant® induced balanced, long‐lasting immune responses that resulted in a strong anti‐tumor activity. A direct comparison to Poly(I:C) showed superior efficacy of our adjuvant to enhance antigen‐specific multifunctional CD8+ T‐cell responses and mediate anti‐tumor responses induced by peptide derived from HPV‐16 E7 protein in the syngeneic TC‐1 tumor, a murine model of human HPV‐induced cervical cancer. Moreover, the adjuvant was able to induce functional memory responses that mediated complete tumor remission. Despite its remarkable immunostimulatory activity, our RNA‐based adjuvant exhibited an excellent pre‐clinical safety profile. It acted only locally at the injection site where it elicited a transient but strong up‐regulation of pro‐inflammatory and anti‐viral cytokines as well as cytoplasmic RNA sensors without systemic cytokine release. This was followed by the activation of immune cells in the draining lymph nodes. Our data indicate that our RNA‐based adjuvant is a safe and potent immunostimulator that may profoundly improve the efficacy of a variety of cancer vaccines.

A CD14 Domain with Lipopolysaccharide‐Binding and ‐Neutralizing Activity

The interaction of lipopolysaccharide with CD14 plays a key role in signaling that activates an early defense against pathogens but also contributes to the development of sepsis and septic shock. Here we have mapped the entire 356‐amino‐acid protein with synthetic 20‐amino‐acid peptides and have identified a new lipopolysaccharide‐binding domain with a strong LPS‐neutralizing activity. Moreover, analysis of the structure–activity relationship of this peptide, which corresponds to amino acids 81–100 of human CD14, revealed that leucines 87, 91, and 94 are essential for these activities. The functional relevance of these residues was confirmed by cellular expression of mutant CD14 proteins that are no longer able to bind LPS. Furthermore, the peptide provided a basis for the generation of highly soluble analogues with stronger lipopolysaccharide‐neutralizing activity.

The activity of lipopeptide TLR2 agonists critically depends on the presence of solubilizers.

Lipoproteins activate cells of the innate immune system via heteromers of Toll‐like receptor (TLR) 2 with either TLR1 or TLR6. In spite of progress in understanding TLR‐dependent signal transduction and the pathophysiological relevance of TLR2, the molecular basis of ligand recognition by this receptor is poorly defined. Here, we show that the bioactivity of lipopeptides (LP) critically depends on the dilution protocol and especially the presence of proteins or detergents acting as solubilizers. Fluorescence correlation spectroscopy of fluorescently labeled analogs of synthetic LP revealed that the LP form aggregates in solution. Dilution into protein‐ and serum‐free buffers led to a complete loss of activity due to formation of large and highly heterogeneous aggregates. When dimethylsulfoxide stock solutions were diluted into BSA or serum‐containing buffers particles of strongly reduced size were obtained. For some LP, an intermediary dilution step either with tert.‐butyl alcohol/H2O (4:1) or with octyl‐β‐D‐glucopyranoside further increased activity. For a panel of LP exhibiting very different activities when diluted directly into protein‐containing solutions, introduction of this dilution step resulted in comparable bioactivities. These results demonstrate the significance of solubilizing agents for the bioactivity of LP and are highly relevant for analyzing structure‐activity relationships of LP‐dependent TLR2 activation.

Chemolabile cellular microarrays for screening small molecules and peptides

Microarrays that mediate the uptake of small molecules into living cells are described. Tissue culture cells were seeded onto glass substrates functionalized locally with fluorescently labelled test substances. In order to enable a localized transferof substances after contact of cells with the substrate, substances were immobilized on the surface either by non-covalent interactions or chemolabile linker groups. These chemolabile linker groups were incorporated into covalently immobilized compounds. Different ester linkages were evaluated as chemolabile linker groups. As model compounds, esters of the carboxy group of a cysteine with the hydroxy groups of carboxyfluorescein-labelled serine amide and tyrosine amide residues or the thiol group of another fluorescein-labelled cysteine amide were generated. Covalent immobilization occurred on maleimide-functionalized glass cover slips. The surface functionalization and release kinetics were assessed by confocal laser scanning microscopy. The fastest release was obtained for the phenolic tyrosine ester. Alternatively, fluorescently labelled peptides were immobilized by non-covalent interactions on glass and on a hydrogel matrix. In order to increase the efficiency of cellular uptake, peptides were N-terminally extended with a cell-penetrating peptide. Uptake of these peptides into cells was confined to the functionalized spots, and was specificfor peptides extended with the cell-penetrating peptide.

RNAdjuvant®, a novel, highly-potent RNA-based adjuvant, combines strong immunostimulatory capacities with a favorable safety profile.

Meeting abstracts Purified recombinant proteins and peptides, which are currently under development in various anti-cancer vaccination approaches, lack sufficient immunogenicity. Therefore, potent adjuvants are needed to induce strong and persistent anti-tumor immunity. However, currently only few