TLR7 and TLR8 as targets in cancer therapy (1)

Toll-like receptors 7 (TLR7) and TLR8 are phylogenetically and structurally closely related members of the TLR family; together with TLR9 they constitute one of the six major TLR clades. With the exception of a small proportion of TLR8 which is expressed at the cell surface, both TLR7 and TLR8 are localized intracellularly to endosomal membranes, similar to TLR3 and TLR9. Both TLR7 and TLR8 have been identified as natural receptors for single-stranded RNA, and they are thought to act as potent activators of innate immune responses upon viral infections. Certain siRNA sequences may even stimulate TLR7/8 independently of their GU content suggesting specific sequences recognized by TLR7/8.

Imiquimod, which is a nucleoside analogue, activates preferentially TLR7; its agonistic activity at TLR8 appears to be much weaker. Resiquimod is a selective ligand for TLR7 in the murine and for TLR7 and TLR8 in the human system. Resiquimod induces more pronounced cytokine secretion, macrophage activation and enhancement of cellular immunity as compared to imiquimod. Gardiquimod induces activation of nuclear factor (NF)-κB in cells expressing human or murine TLR7. At high concentrations, gardiquimod also activates TLR8. CL075 preferentially stimulates TLR8 in human PBMC. Based upon studies using transfected HEK293 cells, concentrations of CL075 required for activation of TLR7 are approximately 10-fold higher than those needed for TLR8 stimulation. CL097 predominantly activates TLR7. Loxoribine is a guanosine analog derivatized at positions C8 and N7. The activation of the innate immune system through loxoribine is, similarly to imiquimod, largely restricted to TLR7.

The specific TLR7 agonist (3M-001) primarily activated plasmacytoid dendritic cells, B cells and monocytes, while a TLR8 agonist (3M-002) induced cytokine production by myeloid dendritic cells, monocytes and monocyte-derived dendritic cells. Accordingly, the TLR7 agonist preferentially induced secretion of IFNα and IFN-regulated chemokines, such as I-TAC (CXCL11) and IP-10 (CXCL10), while the TLR8 agonist predominantly induced proinflammatory cytokines and chemokines including TNFα, IL-12 and MIP-1α (CCL3). The activity of imidazoquinoline agonists at TLR7 and TLR8, respectively, can be modulated by modified oligodeoxynucleotides. Interestingly, such treatments inhibit TLR7 while they enhance TLR8 signaling induced by the respective agonists. The functionality of murine TLR8 has first been demonstrated by using a small-molecule TLR8 agonist (3M-002) together with polyT oligodeoxynucleotides. Antitumoral immune responses is the differential triggering of NK cell activity by TLR8 agonists showing more pronounced indirect NK cell activation as compared to TLR7 agonists.

Imiquimod concentrations of up to 5 μg ml−1 elicited a robust proinflammatory response by dendritic cells. A number of additional imidazoquinolines have been tested in vitro for their capacity to induce cytokine and chemokine production, and for some species effective concentrations of as low as 0.05 μg ml−1. Imiquimod induces expression of proinflammatory cytokines including IFNα, TNFα, IL-2, -6, -8, -12, G-CSF and GM-CSF, as well as chemokines such as CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL2 (MCP-1). The net result of the imiquimod-induced cytokine production is a Th1-weighted cellular immune response, while Th2 signaling pathways appear to be suppressed.

Imiquimod-enhanced activation and immigration of cytotoxic T cells has been observed in vaccination studies with melanoma antigens. For example, imiquimod strongly enhances antigen-specific activation of antitumoral T cells and augments protective cellular antitumoral immunity against melanoma cells following vaccination with Listeria.

Under experimental conditions, there is some evidence suggesting that imiquimod can enhance antibody production by B cells, a function that is typically governed through a Th2-dominated microenvironment. Imiquimod and related compounds exert a direct stimulatory effect on antibody production inasmuch as they mimic CD40-dependent signals, which are normally sent by T cells. In addition, imidazoquinolines can stimulate the proliferation of cultured B cells, even in the absence of other immunocytes, although the increase in B-cell proliferation was more pronounced when preactivated cultures were exposed to imiquimod. Imiquimod can activate NF-κB and increase expression of some proinflammatory cytokines (IL-6, IL-8, TNFα and IL-1β) in TLR-7- and TLR-8-negative cell lines.

1. M. P. Schön, M. Schön, TLR7 and TLR8 as targets in cancer therapy. Oncogene. 27, 190–199 (2008).