Toll like receptors (TLRs) are a class of pattern recognition receptors that play a bridge role in the innate immunity and adaptive immunity. The activated TLRs not only induce inflammatory responses, but also lead to the development of antigen specific immunity. TLR7, a member of TLR family, is an intracellular receptor expressed on the membrane of endosomes, and its powerful immune stimulatory action can be potentially used in the anti-tumor therapy. This article reviews the anti-tumor activities of the agonists of TLR7, which are frequently applied in the preclinical and clinical investigations.

Background: Various tumors are insensitive to immune checkpoint blockade (ICB) therapy. Toll-like receptors (TLRs) establish the link between innate and adaptive immunity, which can assist T-cell activation and serve as promising targets for combination to enhance ICB therapy. Here, we aimed to improve efficacy for anti-programmed death ligand 1 (PD-L1) therapy by developing a PD-L1/TLR7 dual-targeting nanobody-drug conjugate (NDC), based on the PD-L1 nanobodies and TLR7 agonist we developed. Methods: PD-L1 nanobodies were obtained by phage display screening and identified through T-cell activation bioassay, in vivo imaging and quantitative biodistribution study. Immune activation and PD-L1-inducing of TLR7 agonists were evaluated in diverse innate cell models. We constructed PD-L1/TLR7 dual-targeting NDCs by chemically coupling PD-L1 nanobodies and TLR7 agonists. The antitumor effect was evaluated via several murine or humanized solid tumor models. Immunophenotyping, immune cell depletion, tumor rechallenge, RNA sequencing and PD-L1-deficient models were combined to determine the mechanism for NDCs function. The dynamics of the in vivo behaviors of NDCs were assessed based on multiorgan changes in PD-L1 levels. Results: The screened PD-L1 nanobodies were characterized as tumor-targeting and alleviated T-cell immunosuppression. The TLR7 agonists induced broad innate immune responses and intratumoral PD-L1 expression on antigen-presenting cells (APCs), and its antitumor effect was dependent on intratumoral delivery. The combination of TLR7 agonists and PD-L1 nanobodies activated both innate and adaptive immunity and upregulated PD-L1-related signaling pathways. After coupling to form dual-targeting NDCs, TLR7 agonists and PD-L1 nanobodies exerted synergistic antitumor effects and safety in either ‘hot’ or ‘cold’ tumor and early or advanced tumor models, reshaped the tumor immune microenvironment and induced antitumor immune memory. CD8⁺ T cells and natural killer cells were the main effector cells for NDCs to function. NDCs can promote PD-L1 expression on intratumoral APCs and tumor cells, and subsequently achieve targeted enrichment in tumors. Moreover, the efficacy of NDCs is biased toward dependence on host expression of PD-L1. Conclusions: The novel PD-L1/TLR7 dual-targeting NDC exhibited potent efficacy against heterogeneous tumors through orchestrating innate and adaptive immunity, which could act as a promising strategy to improve ICB therapy and shows prospects for clinical development.

Conventional chemotherapy and radiotherapy for the treatment of lymphoma have notable drawbacks, and passive immunotherapy using a monoclonal antibody is restricted to CD20-positive B cell lymphoma. Therefore, new treatment types are urgently required, especially for T cell lymphoma. One type of new antitumour therapy is the use of active immunotherapeutic agents, such as agonists of the Toll-like receptors (TLRs), which facilitate the induction of prolonged antitumour immune responses. We have synthesised a novel TLR7 agonist called SZU-101 and investigated the systemic antitumour effect on a murine model of T cell lymphoma in vivo. Here, we report that the intratumoural administration of SZU-101 enhanced the effectiveness of a conventionally used chemotherapeutic agent, doxorubicin (DOX). SZU-101 administration improved tumour clearance in a murine model of T cell lymphoma. The novel combination of intratumourally administered SZU-101 and DOX generated strong cytokine production and enhanced the cytotoxic T lymphocyte response, leading to the eradication of both local and distant tumours in tumour-bearing mice. These findings suggested that combined active immunotherapy can be developed as a promising treatment for T cell lymphoma, which may further improve the effectiveness of the current standard cyclophosphamide, DOX, vincristine and prednisone (CHOP) therapy.

A combination of platinum drugs with immunotherapy has shown promising anticancer effects, especially in the drug resistance cancer model. Herein, a new type of immunochemotherapeutic was designed by tethering the toll-like receptor 7 (TLR7) agonist on the axial position of oxaliplatin-based platinum(IV) prodrug. The prodrug simultaneously induced immunogenic cell death of 4T1 cancer cells to initiate an immune response and activate dendritic cells (DCs) to secrete proinflammatory cytokines including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-12, to further enhance the adaptive immunity. The prodrug exhibited better in vivo anticancer effects than oxaliplatin in the 4T1 allograft mouse model, a later stage breast cancer model, which showed poor response to traditional chemotherapy. Mechanism studies revealed that enhanced activation of cytotoxic T cells within tumor contribute to the high in vivo anticancer efficiency of the prodrug. Moreover, the prodrug displayed much lower cytotoxicity to DCs compared with oxaliplatin, indicating its safety to normal cells. These results highlight the potential of the conjugation of TLR7 agonist with oxaliplatin-based Pt(IV) prodrug as an effective anticancer agent to overcome the toxic side effects and drug resistance of traditional platinum chemotherapy.

Vaccines play increasingly important roles in cancer treatment due to their advantages of effective targeting and few side effects. Our laboratory has attempted to construct vaccines by conjugating TLR7 agonists with tumor-associated antigens. Furthermore, immunochemotherapy has recently become an appealing approach to cancer therapy. 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent, can reportedly potently and selectively kill tumor-associated MDSCs in vivo. Gastric cancer vaccines were synthesized by the covalent attachment of our TLR7 agonist with the gastric cancer antigen MG7-Ag tetra-epitope, leading to T7 − ML (linear tetra-epitope) and T7 − MB (branched tetra-epitope). Cytokines induced by the vaccines in vitro were assessed by ELISA. A tumor challenge model was created by treating BALB/c mice on either a prophylactic or therapeutic vaccination schedule. 5-FU was simultaneously applied to mice in the combination treatment group. CTL and ADCC activities were determined by the LDH method, while CD3+/CD8+, CD3+/CD4+ T cells and MDSCs were evaluated by flow cytometry. In vitro, rapid TNF-α and IL-12 inductions occurred in BMDCs treated with the vaccines. In vivo, among all the vaccines tested, T7 − MB most effectively reduced EAC tumor burdens and induced CTLs, antibodies and ADCC activity in BALB/c mice. Immunization with T7 − MB in combination with 5-FU chemotherapy reduced tumor sizes and extended long-term survival rates, mainly by improving T cell responses, including CTLs, CD3+/CD8+ and CD3+/CD4+ T cells. 5-FU also enhanced the T7 − MB efficiency by reversing immunosuppressive factors, i.e., MDSCs, which could not be validly inhibited by the vaccines alone. In addition, T7 − MB repressed tumor growth and immune tolerance when the therapeutic schedule was used, although the effects were weaker than those achieved with either T7 − MB alone or in combination with 5-FU on the prophylactic schedule. A novel effective gastric cancer vaccine was constructed, and the importance of branched multiple antigen peptides and chemical conjugation to vaccine design were confirmed. The synergistic effects and mechanisms of T7 − MB and 5-FU were also established, observing mainly T cell activation and MDSC inhibition.

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