BackgroundLigands for the Toll-like receptor (TLR) family can induce activation of cells of the innate immune system and are widely studied for their potential to enhance adaptive immunity. Conjugation of TLR2-ligand Pam3CSK4 to synthetic long peptides (SLPs) was shown to strongly enhance the induction of antitumor immunity. To further improve cancer vaccination, we have previously shown that the novel TLR2-L Amplivant (AV), a modified Pam3CSK4, potentiates the maturation effects on murine DCs. In the current study, we further assessed the immunological properties of AV.MethodsNaïve mice were vaccinated with a conjugate of either Pam3CSK4 or AV and an SLP to assess specific T cell priming efficiency in vivo. The potency of AV and Pam3CSK4, either as free compounds or conjugated to different SLPs, to mature murine DCs was compared by stimulating murine dendritic cells overnight followed by ELISA and flow cytometry analysis. Murine tumor experiments were carried out by vaccinating mice carrying established HPV16 E6 and E7-expressing tumors and subsequently analyzing myeloid and lymphoid cells infiltrating the tumor microenvironment. Furthermore, tumor outgrowth after vaccination was monitored to enable comparison of the efficiency to induce antitumor immunity by Pam3CSK-SLP and AV-SLP conjugates. To enhance therapeutic efficacy, AV-SLP conjugate vaccination was combined with ablative therapies to assess whether synergism between such therapies would occur.ResultsSLPs conjugated to AV induce stronger DC maturation, in vivo T cell priming and antitumor immunity compared to conjugates with Pam3CSK4. Interestingly, AV-SLP conjugates modulate the macrophage populations in the tumor microenvironment, correlating with a therapeutic effect in an aggressive murine tumor model. The potency of AV-SLP conjugates in cancer vaccination operates optimally in combination with chemotherapy or photodynamic therapy.ConclusionThese data allow further optimization of vaccination-based immunotherapy of cancer by use of the improved TLR2-ligand Amplivant.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0455-2) contains supplementary material, which is available to authorized users.

New analogues (UPam) of triacylated lipopeptide Pam3CysSK4, a popular agonist of Toll-like receptor 2 (TLR2), were designed making use of the cocrystal structure of a TLR2 heterodimer (with TLR1) with Pam3CysSK4. Twenty-two UPam derivatives that feature an N-tetradecylcarbamyl chain to mimic the native N-palmitoyl moiety and various small amino acids residues at the penultimate N-terminal position were prepared via solid-phase synthesis. In vitro evaluation of immunostimulatory properties revealed new potent TLR2 ligands.

Muramyl dipeptide (MDP) is the smallest peptidoglycan fragment capable of triggering the innate immune system through interaction with the intracellular NOD2 receptor. To develop synthetic vaccine modalities composed of an antigenic entity (typically a small peptide) and a molecular adjuvant with well-defined activity, we previously assembled covalent MDP-antigen conjugates. Although these were found to be capable of stimulating the NOD2 receptor and were processed by dendritic cells (DCs) leading to effective antigen presentation, DC maturation--required for an apt immune response--could not be achieved with these conjugates. To improve the efficacy of these vaccine modalities, we equipped the MDP moiety with lipophilic tails, well-known modifications to enhance the immune-stimulatory activity of MDPs. Herein we report the design and synthesis of a lipophilic MDP-antigen conjugate and show that it is a promising vaccine modality capable of stimulating the NOD2 receptor, maturing DCs, and delivering antigen cargo into the MHC-I cross-presentation pathway.

SummaryThe covalent attachment of an innate immune system stimulating agent to an antigen can provide active vaccine modalities capable of eliciting a potent immune response against the incorporated antigen. Here we describe the design, automated synthesis and immunological evaluation of a set of four muramyl dipeptide–peptide antigen conjugates. Muramyl dipeptide (MDP) represents a well-known ligand for the intracellular NOD2 receptor and our study shows that covalently linking an MDP-moiety to an antigenic peptide can lead to a construct that is capable of stimulating the NOD2 receptor if the ligand is attached at the anomeric center of the muramic acid. The constructs can be processed by dendritic cells (DCs) and the conjugation does not adversely affect the presentation of the incorporated SIINFEKL epitope on MHC-I molecules. However, stimulation of the NOD2 receptor in DCs was not sufficient to provide a strong immunostimulatory signal.

Simultaneous triggering of Toll-like receptors (TLRs) and NOD-like receptors (NLRs) has previously been shown to synergistically activate monocytes, dendritic cells, and macrophages. We applied these properties in a T-cell vaccine setting by conjugating the NOD2-ligand muramyl-dipeptide (MDP) and TLR2-ligand Pam3CSK4 to a synthetic peptide derived from a model antigen. Stimulation of human DCs with the MDP-peptide-Pam3CSK4 conjugate led to a strongly increased secretion of pro-inflammatory and Th1-type cytokines and chemokines. We further show that the conjugated ligands retain their ability to trigger their respective receptors, while even improving NOD2-triggering. Also, activation of murine DCs was enhanced by the dual triggering, ultimately leading to effective induction of vaccine-specific T cells expressing IFNγ, IL-2, and TNFα. Together, these data indicate that the dual MDP-SLP-Pam3CSK4 conjugate constitutes a chemically well-defined vaccine approach that holds promise for the use in the treatment of virus infections and cancer.