Anti-tumor Activity of Toll-Like Receptor 7 Agonists

Toll-like receptors (TLRs) are a class of pattern recognition receptors that play a bridging role in innate immunity and adaptive immunity. The activated TLRs not only induce inflammatory responses, but also elicit the development of antigen specific immunity. TLR7, a member of TLR family, is an intracellular receptor expressed on the membrane of endosomes. TLR7 can be triggered not only by ssRNA during viral infections, but also by immune modifiers that share a similar structure to nucleosides. Its powerful immune stimulatory action can be potentially used in the anti-tumor therapy. This article reviewed the anti-tumor activity and mechanism of TLR7 agonists that are frequently applied in preclinical and clinical investigations, and mainly focused on small synthetic molecules, including imiquimod, resiquimod, gardiquimod, and 852A, etc.


INTRODUCTION
Toll-like receptors (TLRs) are a class of pattern recognition receptors that play a bridging role in innate immunity and adaptive immunity (O'Neill et al., 2013). TLRs can recognize both pathogen-associated molecular patterns and damage-associated molecular patterns such as lipopolysaccharide and free nucleic acids (Piccinini and Midwood, 2010). Normally, TLRs are expressed by macrophages, dendritic cells, natural killer (NK) cells and epithelial cells. Some TLRs are expressed in the intracellular endosomes (TLR3,7,8,and 9), while others are localized on the plasmalemma (TLR1, 2, 4, 5, 6, 10, and 11;Hennessy et al., 2010). TLRs are activated by diverse agonists, e.g., TLR4 by lipopolysaccharide, and TLR3, 7 and 9 by nucleic acids ( Table 1). The binding of a TLR and its agonist generates an immune response, important for therapeutic research, including research on anti-cancer therapy. TLR agonist-based cancer immunotherapy has been used in preclinical and clinical investigations (Vacchelli et al., 2013). Most of the TLR agonists are clinically tolerated and biologically active, but some common adverse events were observed. The development of many TLR agonists has been discontinued in consequence of lacking efficacy in phase III trial . Among TLRs, TLR7 is an intracellular receptor expressed on endosomal membranes. TLR7 is closely related to TLR8, which also recognizes nucleosides and nucleotides from intracellular pathogens. There are two ligand-binding sites in TLR7. The first site for binding of small ligands is conserved in both TLR7 and TLR8. The second site differs from that  of TLR8, and is used to bind with ssRNA to enhance activation of the first site (Maeda and Akira, 2016). Activation of TLR7 can induce Type 1 interferon and inflammatory response, therefore targeting TLR7 is a promising strategy for both antiviral and antitumor therapy. This article aims to review the anti-tumor activity of TLR7 agonists with a focus on small synthetic molecules.

IMIQUIMOD
Imiquimod, also called Aldara or R-837, is an immune response modifier acting as a TLR7 agonist. It has been approved by Food and Drug Administration (FDA) as a therapeutic agent for basal cell carcinoma and genital warts . The structure of imiquimod is similar to adenosine nucleoside and it can interact with adenosine receptors. Imiquimod has now been well studied and more and more research is focusing on its anti-tumor activities. TLR7 is highly expressed in oral squamous cell carcinoma cells. The growth of these cells can be significantly inhibited and apoptosis through mitochondriadependent pathway brought about by treatment with imiquimod. (Ahn et al., 2012). From another aspect, effector T cells from imiquimod-treated squamous cell carcinoma cells generate more IFN-γ and less IL-10 compared with untreated cells (Huang et al., 2009). Imiquimod inhibits the proliferation and also arrests the cell cycle in both murine and human prostate cancer cells . However, the anti-tumor activities of imiquimod can be counteracted by immunosuppressive cytokines and other molecules, such as IL-10, indoleamine 2,3-dioxygenase (IDO) and induced nitric oxide synthase. IDO expression is upregulated by imiquimod. Therefore, different kinds of combinational therapy with imiquimod have been used to overcome the shortcoming of using imiquimod alone. Ito et al. (2015a) found that combining imiquimod with 1-methyl-D-tryptophan, an IDO inhibitor, largely inhibited the growth of tumor cells, and enhanced the efficacy of imiquimod through induction of Th1 response. Blocking of IL-10 and nitric oxide synthase was also applied to combination therapy using imiquimod (Lu et al., 2010;Ito et al., 2015b). In a combined chemotherapy in phase II clinical trial, breast cancer patients were treated with imiquimod plus albumin-bound paclitaxel, disease regression was induced and 92% of adverse events were grade 1 and grade 2. However, the responses were short-lived (Salazar et al., 2017). Using TLR agonists as chemoadjuvants decreased the possibility of adverse events and enhanced the efficiency of chemical agents with a diminished dosage, but survival prospects were still modest (Ding et al., 2017).
Imiquimod is slightly soluble in common solvents and insoluble in water. Hence poloxamer polymer was added to prolong local contact and lessen systemic absorption of imiquimod. Incorporating 2-(hydroxypropyl)-β-cyclodextrin in the formulation should augment the physical stability, and a clear homogeneous solution was produced. This kind of combination enhanced chemokine induction and showed anti-tumor effects in an orthotopic mouse model of bladder cancer (Hayashi et al., 2010). Local imiquimod treatment induced a systemic antigen-specific CD8 response, but did not prevent the growth of distal tumor because of a lack of CD4 T cell response. Combining local imiquimod with anti-CD40 therapy reinforced the local response, and upregulated the ratio of regression of distal tumor (Broomfield et al., 2009;Dewan et al., 2012) found that imiquimod inhibited the growth of cutaneous breast cancer cells by a CD8 dependent mechanism, but did not cause complete tumor regression. In addition, the effects of imiquimod were abolished with depletion of CD8+ T cells. Synergistic effects of local radiotherapy and imiquimod could rectify this situation, and a low dose of cyclophosphamide further enhanced these effects, and reduced recurrence by inducing protective immunological memory.
Imiquimod is an FDA-approved TLR agonist that has aroused considerable clinical research interest, and showed promising results in clinical studies. In seven imiquimod treated BCC patients, complete histopathological tumor clearance was observed 6 weeks after initiation of treatments and no signs of recurrence were detected. Most importantly, systemic side effects such as myalgia, lymphadenopathy were indiscernible during the trial (Love et al., 2016). In patients with genital warts, the serum concentrations were low after daily treatment of imiquimod. There were no serious adverse events, indicating the safety of imiquimod . Moreover, imiquimod can promote a pro-immunogenic tumor environment. Melanoma metastases often fail to respond to immune therapies because of the lack of T cells. Based on immune activation, imiquimod could be an efficacious therapeutic agent against melanoma (Narayan et al., 2012). Four patients, treated with imiquimod daily and immunized with a vaccine consisting of a tetanus toxoid-derived helper peptide and 12 melanom peptides, showed increased expression of cytokines and chemokines as well as CD8+T cell infiltrates (Mauldin et al., 2016).

RESIQUIMOD AND GARDIQUIMOD
Both resiquimod and gardiquimod have an imidazoquinoline structure, share a similar structure with imiquimod, but have more potential properties than imiquimod.
Targeting tumor angiogenesis has become a prospective strategy for treating cancer in view of the important role of angiogenesis in tumor proliferation. However, a lot of studies have pointed out that the use of anti-angiogenic agent may lead to immunosuppression. So combination therapy is necessitated. Resiquimod exhibited a robust anti-tumor activity in a mouse breast cancer model, combining resiquimod with sunitinib, an antineoplastic agent, largely inhibited the growth of breast cancer cells, and attenuated the immunosuppressive effects of sunitinib (Yin et al., 2015). Cutaneous T-cell lymphoma is malignant tumor of the immune system caused by a mutation of T cells. The malignant T cells migrate to the skin and cause lesions. There is no cure in addition to preventing transplantation. Twelve patients were treated with topical resiquimod gel in a phase I trial. The data revealed that 75% of the patients had improved lesions and 30% of the patients had all lesions cleared. T-cell receptor sequencing demonstrated a decrease of malignant T cells in 90% of the patients and complete elimination in 30% of patients (Rook et al., 2015). It was reported that resiquimod could induce apoptosis of acute myeloid leukemia cells and upregulate the expression of MHC molecules on membranes of acute myeloid leukemia cells. Furthermore, the production of cytokines IL-6, IFN-γ and TNF-α was distinctly elevated (Smits et al., 2010). Resiquimod in combination with radiation therapy, induced expansion of antigen-specific CD8+T cells and prolonged the survival of T cell lymphoma tumor-bearing mice . Resiquimod also has numerous applications as immune adjuvants. Upon treatment with NY-ESO-1, a widely used tumor antigen for vaccination, and imiquimod, CD4+T cell responses but not CD8+T cell responses could be observed in melanoma patients. Thus, Sabado et al. combined another TLR7 agonist, resiquimod, with NY-ESO 1 in treating patients with resected high risk melanoma, and found that CD8+T cell response was increased in a small subset of patients (Sabado et al., 2015). Human papillomavirus (HPV) type 16 is associated with the generation of cervical cancer. Nevertheless, Langerhans cells which serve as antigen presenting cells in the viral infection failed to induce T cell immune response when exposed to HPV16. Resiquimod activated Langerhans cells exposed to HPV16 and induced a specific CD8+ T cell response (Fahey et al., 2009). Oral administration of 0.01 mg/kg resiquimod was tolerated, but serious adverse events were observed at 0.02 mg/kg in patients with hepatitis C virus infection. The 0.02 mg/kg dose of resiquimod leads to IFN-like side effects. Therefore, further studies are necessary to demonstrate the efficacy and safety of TLR7 agonists (Pockros et al., 2007).
Gardiquimod has also been used in cancer therapy. It exhibited a series of potential benefits in oncotherapy, inhibition of cell proliferation, triggering of apoptosis, and suppression of metastasis etc. (Ma et al., 2010;Weber et al., 2013;Zou et al., 2015). These results suggest that the imidazoquinoline family is promising for application in clinical cancer therapy.

852A
852A, a TLR7-specific agonist, is more potent and selective than imiquimod. Harrison et al. (2007) evaluated the bioavailability, pharmacokinetics and pharmacodynamics of 852A in a phase I trial by employing different ways of drug delivery. Eighteen healthy adult volunteers were enrolled in the trial. Pharmacokinetic parameters revealed that the subcutaneous route with a bioavailability of 80.5 ± 12.8% was a promising route of administration for subsequent evaluation. Serum concentrations of TNF-α and C-reactive protein were upregulated. Since 852A has a potent immunostimulating function, Geller et al. (2010) explored the anti-tumor activities of 852A in patients with recurrent ovarian cervix and breast cancers. Fifteen patients received 852A subcutaneously for 12 weeks. Sustained tolerability was observed and clinical benefit was modest. The same prolonged subcutaneous administration has also been applied to the treatment of recurrent hematologic malignancies, and demonstrated measurable immune activation (Weigel et al., 2012).
The anti-melanoma activity of 852A was explored in a phase II trial. Patients with chemotherapy-refractory metastatic melanoma received an intravenous injection of 852A which caused systemic immune activation and disease stabilization, even though objective clinical responses were not observed (Dummer et al., 2008). 852A is regarded as a perfect tool for studying the molecular processes of TLR7 in plasmacytoid dendritic cells (pDC). It stimulates pDC for the production of IFNα, and the inhibition of cell proliferaton also depends on pDC and type 1 IFN (Inglefield et al., 2008).

OTHER AGONISTS
Numerous studies have focused on synthesizing a variety of small molecules to serve as agonists of TLR7. Loxoribine (7allyl-8-oxoguanosine) enhanced NK cells activity and induced production of cytokines such as IFNs. It is expected to be helpful to cancer therapy clinically (Agarwala et al., 2000). Bropirimine (2-amin-5-bromo-6-phenyl-4(3)-pyrimidinone) is an orally administered modulator used against renal cell carcinoma in the clinical setting and could induce production of cytokines including IFN-α (Sarosdy et al., 2005). GS-9620 [8-(3-(pyrrolidin-1-ylmethyl) benzyl)-4-amino-2-butoxy-7,8-dihydropteridin-6(5H)-one] is an eligible agonist of TLR7 for the treatment of chronic hepatitis B viral infection. Additionally, oral administration of GS-9620 manifested an antiviral activity without the adverse effects characteristic of systemic response to IFN-α. (Lanford et al., 2013;Fosdick et al., 2014;Bam et al., 2017). 3M-052 is an insoluble injectable TLR7/8 agonist. Intratumoral injection of 3M-052 induced systemic anti-tumor activity and inhibited both local and distal tumor growth in mice bearing wild type B16.F10 melanoma. 3M-052 combined with a checkpoint inhibitor could considerably enhance the effects (Singh et al., 2014). Similarly, 3M-011 boosted the antigen-presenting activities of DC as an adjuvant to radiation therapy. This kind of combination therapy induced local and systemic responses in pancreatic cancer mouse models (Scholch et al., 2015). DSR-6434 has higher water solubility and is more potent toward TLR7 than 852A as mentioned above. Systemic administration of DSR-6434 may reinforce the effect of radiation therapy of cancer in mouse models. However, upon administration twice a week, TLR tolerance emerged and no antitumor activity was observed compared with administration once a week, suggesting that activation of DSR-6434 occurs in a dosedependent manner (Nakamura et al., 2013;Adlard et al., 2014;Koga-Yamakawa et al., 2015). Analogous molecules such as DSR-29133 also have similar potential and the anti-tumor effects can be fortified by combining with low-dose fractionated radiation therapy (Dovedi et al., 2016). Furthermore, SC1, a small molecule agonist of TLR7, has been demonstrated to stimulate NK cells and therefore mediate efficient immune responses, and showed an effective anti-metastatic activity in vivo. More specifically, mice bearing NK cell sensitive RMA-S lymphoma were cured by repetitive subcutaneous injections of SC1, and no toxicity or recurrence was observed (Hamm et al., 2009;Wiedemann et al., 2016).
In addition, combining a TLR7 agonist with doxorubicin could be a promising treatment for T cell lymphoma (Zhu et al., 2015). The novel TLR7 agonist SZU-101 which was synthesized in ShenZhen University was also applied to immune adjuvant and acquired prospective outcomes Diao et al., 2016). There are also many TLR7 agonists attracting researchers' interest: SM-276001 and SM -360320 are selective TLR7 agonist, and SM-360320 can synergize with DNA vaccines targeting CEA colon cancer and HER2 breast cancer Koga-Yamakawa et al., 2013).

ANTI-TUMOR MECHANISM
After binding ligands, TLRs change structure for the recruitment of myeloid differentiation primary-response protein 88(MYD88) and TIR-domain-containing adaptor-inducing interferon-β (TRIF). The pathways are MYD88-dependent and MYD88independent. MyD88 has an amino (N)-terminal death domain (DD), a carboxy (C)-terminal TIR domain, and an intermediate domain which is crucial for TLR signaling (Zou et al., 2016). In the MYD88-dependent pathway, MYD88 complexes with IL-1R-associated kinases 4 (IRAK4) in turn interact with IRAK1 and IRAK2. This kind of association is based on a DD-DD interaction. IRAK4 leads to phosphorylation of IRAK1 and IRAK2, thus promoting its association with tumor necrosis factor (TNF) R-associated factor 6 (TRAF6). TRAF6 is an E3 ubiquitin ligase which takes part in the phosphorylation of transforming growth factor beta-activated kinase 1 (TAK1). Subsequently, these complexes on the one hand activate nuclear factor kappa B (NF-κB), on the other hand, leading to the translocation of Interferon regulatory factor 7 (IRF7; Figure 1).
TLR7 agonists will become inoperative unless delivered to endosomal vesicles, where the receptor resides. Conjugation of a TLR7 agonist to phospholipids, which is widely applied in drug delivery, can improve its bioavailability as well as the immune response. However, not all of the delivery molecules can induce a comparable response. TLR7 agonists conjugated to mouse serum albumin induced a small quantity of Type 1 interferons. Probably, TLR7 agonists and phospholipid conjugation could facilitate the uptake by pDC and enhance persistence of ligands in endosomal vesicles (Chan et al., 2009). PDC is the major source of Type 1 interferon. It was reported that pDC recognition of pathogens was mainly mediated by TLR7 and TLR9 pathway. Most of the TLR7 agonists have no direct cytotoxic effects on tumor cells. It was reported that deficiency of NK cells might interdict the effects of TLR7 agonists. 3M-011 lost its anti-tumor property in NK1.1-immunodepleted mice, demonstrating the crucial role of NK cells in anti-tumor activities (Dumitru et al., 2009).
There was another discovery which demonstrated that TLR7 activation by imiquimod and resiquimod could induce apoptosis of cancer cells in an assay involving Annexin V-staining. Further study revealed that expression of Bcl-2 was downregulated and cleavage of caspase-3, caspase-7 was upregulated in imiquimod-treated cells (Smits et al., 2010), suggesting that the apoptosis-inducing effects of imiquimod were involved in a caspase-dependent mitochondrial pathway. Futhermore, FIGURE 1 | The signaling pathways of TLRs include MYD-88 independent and MYD-88 dependent pathways. Activation of TLR3 and TLR4 rely on MYD88 independent pathway which can activate IRF3 and IRF7, together leading to the induction of Type 1 IFNs. MYD-88 dependent pathway ultimately activates NF-κB and IRF7, inducing secretion of IFNs and some pro-inflammatory cytokines. Stimulation of TLR also activates the apoptosis pathway and PI3K/AKT pathway. TRIF, Toll-like receptor adapter molecule; TRAF6, TNF receptor-associated factor 6; IRAK, Interleukin-1 receptor-associated kinase; NF-κB, Nuclear factor κB; IFN, Interferon. imiquimod induced reactive oxygen species production to stimulate ATM/ATR pathways and lead to p53-dependent apoptosis in the skin basal cell carcinoma cells . However, there were no available studies testifying that MYD88 pathway activates p53 expression. The ability of TLR signaling pathway to cross-talk with other pathways is important for inflammatory response (Figure 1; Brown et al., 2011).
Increased regulatory T cells (Tregs) in cancers are adverse to immune therapy. Activation of some TLRs, e.g., TLR4 may stimulate proliferation of Tregs and promote their suppressive function. TLR7 agonist loxoribin was found to modulate CD4+ T cell proliferation and suppress the activity of Tregs via DCs depending on TLR7 pathway (Wang et al., 2014). Additionally, TLRs activation is associated with some autoimmune disorders, such as systemic lupus erythematosus and autoimmune diabetes. Repeated topical treatment of NOD mice with a TLR7 agonist expedited the onset of autoimmune diabetes (Lee et al., 2011). Moreover, functional TLRs are also expressed on some tumors and play an important role in cancer progression. Activation of tumor TLRs may lead to proliferation of tumor cells and enhancement of tumor invasion (Huang et al., 2005(Huang et al., , 2008Cherfils-Vicini et al., 2010).

CONCLUSION
TLR 7 agonists are small molecules. They stimulate innate immune cells leading to the activation of humoral and cellular immunity, thus engendering a series of anti-tumor activities. The mechanism of action of TLR7 agonists is associated with the MYD88-dependent pathway and caspase-dependent mitochondrial pathway. Further research on the synthesis of currently available TLR7 agonists may not only shed light on their preclinical pharmacological properties, but also on cancer therapy in the clinical setting.

AUTHOR CONTRIBUTIONS
HC and CL conceived the topic and drafted the manuscript. FZ edited the manuscript, and LZ drafted the Table. OS, GJ, and TN supervised the work and revised the paper.