Simultaneous Targeting of Toll- and Nod-Like Receptors Induces Effective Tumor-Specific Immune Responses

Abstract

Toll-like receptor (TLR) ligands are increasingly being used as adjuvants in cancer vaccine trials to harness innate immunity and prime effective antitumor immune responses. Despite some success, enhancing tumor antigen presentation, promoting a protective antitumor response, and overcoming the immunosuppressive tumor microenvironment pose considerable challenges that necessitate further improvements in vaccine design. Here, we show that expression of the TLR ligand flagellin within tumor cells constitutes an effective antitumor vaccination strategy that relies on simultaneous engagement of TLR5 and the Nod-like receptors (NLRs) NLRC4/NAIP5 (neuronal apoptosis inhibitory protein 5) by flagellin along with associative recognition of tumor antigen for optimal antigen presentation to T cells. Although TLR5 signaling was critical for mediating rapid macrophage-dependent clearance of flagellin-expressing tumor cells in vivo, TLR5 and NLRC4/NAIP5 were equally important for priming antitumor CD4(+) and CD8(+) T cells and suppressing tumor growth. Vaccination with irradiated flagellin-expressing tumor cells prevented tumor development, and disrupting flagellin recognition by TLR5 or NLRC4/NAIP5 impaired protective immunization against an existing or subsequent tumor. Our findings delineate a new strategy to induce anticancer immune responses consisting of introducing microbial structures with dual TLR and NLR stimulatory activity into tumor cells. This ensures recognition of tumor-derived antigen within the inflammatory context of microbial recognition and additionally activates both the phagocytic and the cytosolic pathways of innate immune defense against the tumor.

Toll-like receptor-5 agonist, entolimod, suppresses metastasis and induces immunity by stimulating an NK-dendritic-CD8 + T-cell axis

“…In fact, activation of TLR5 by commensal bacteria was shown to drive malignant progression at extramucosal locations through the promotion of inflammation, which dampened antitumor immunity by expanding immunosuppressive networks (65). In contrast, we and others have shown that TLR5 activation has tumor growth suppressive effects and stimulates antitumor immunity (19)(20)(21)(22)(23)(24)(25)(26)53). Earlier studies in our laboratory showed that entolimod displayed a growth-inhibitory effect on a TLR5-expressing mouse xenograft model of human lung adenocarcinoma A549 (23), supporting a direct effect of TLR5 stimulation on tumor growth suppression.…”

“…Studies have shown that the only known natural TLR5 agonist, flagellin, flagellin-expressing Salmonella bacteria, and a pharmacologically optimized flagellin derivative named entolimod (CBLB502) have antitumor effects in several tumor models (19)(20)(21)(22)(23), including mouse models of liver metastases (24)(25)(26). Moreover, systemic administration of TLR5 agonists is uniquely safe because of the restricted pattern of expression of TLR5 (primarily in the gut, liver, and bladder) and the nature of the cytokines induced following TLR5 stimulation.…”

Beyond antigens and adjuvants: formulating future vaccines

“…A few studies to date suggest that such synergies may also be relevant for in vivo responses to vaccination. For example, a cancer vaccine based on irradiated flagellin-and ovalbuminexpressing tumor cells was shown to elicit potent T cell responses in mice, dependent on both TLR5 and the Nod-like receptors NLRC4 and NAIP5 (72). In NHPs, combinations of TLR7/8 and TLR9 agonists enhanced the induction of binding and neutralizing antibody titers against an HIV envelope immunogen (73).…”

Mobiliser l’immunité innée dans le traitement des cancers

“…[29]. Nous avons récemment montré que des cellules tumorales modifiées de telle façon qu'elles expriment la flagelline stimulent efficacement les cellules myéloïdes et génèrent une réponse adaptative spécifique d'antigènes tumoraux [30]. Une telle stratégie pourrait s'avérer prometteuse comme traitement prophylactique ou curatif dans des modèles murins de tumeurs [30].…”

“…Nous avons récemment montré que des cellules tumorales modifiées de telle façon qu'elles expriment la flagelline stimulent efficacement les cellules myéloïdes et génèrent une réponse adaptative spécifique d'antigènes tumoraux [30]. Une telle stratégie pourrait s'avérer prometteuse comme traitement prophylactique ou curatif dans des modèles murins de tumeurs [30]. Elle présente plusieurs avantages : (1) elle permet de cibler deux types de PRR complémentaires en délivrant les ligands correspondants à l'endroit où sont localisés leurs récepteurs ; (2) elle assure la présence du ligand de TLR5 dans le phagosome contenant les cellules tumorales et favorise ainsi la « reconnaissance associative » d'une multitude d'antigènes tumoraux, ne limitant pas cette approche aux seuls cancers dont certains antigènes ont préala-blement été identifiés (Figure 3).…”