Yuwen Diao

Synthesis and Evaluation of Conjugates of Novel TLR7 Inert Ligands as Self-Adjuvanting Immunopotentiators.

During the design and synthesis of a series of 8-hydroxy-2-(2-methoxyethoxy)-adenine derivatives bearing various substituted -RCOOH groups at the 9-position, we identified a TLR7-inert ligand, which does not activate TLR7 signaling pathway. Of interest, the coupling of weakly immunogenic antigens via the -RCOOH group was able to significantly enhance the immunogenicity of the antigens. Herein, an inert ligand, 9-(3-carboxypropyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine (5, GD2), was synthesized and conjugated to 5 different weakly immunogenic antigens (BSA, OVA, MSA, MG7, and thymosin). Compared with the GD2 and the potent agonist UC-1 V150, all conjugates demonstrated potent immunogenicity in vitro and in vivo. All conjugates induced prolonged increases, while UC-1 V150 showed a rapid decline in the levels of proinflammatory cytokines following initial increases. These data indicate that the immunostimulatory activity of TLR7-inert ligands could be amplified and prolonged by conjugation to antigens, thus broadening the potential therapeutic application of these agents.

Conjugation of toll-like receptor-7 agonist to gastric cancer antigen MG7-Ag exerts antitumor effects

AIM To investigate the effects of our tumor vaccines on reversing immune tolerance and generating therapeutic response. METHODS Vaccines were synthesized by solid phase using an Fmoc strategy, where a small molecule toll-like receptor-7 agonist (T7) was conjugated to a monoclonal gastric cancer 7 antigen mono-epitope (T7-MG1) or tri-epitope (T7-MG3). Cytokines were measured in both mouse bone marrow dendritic cells and mouse spleen lymphocytes after exposed to the vaccines. BALB/c mice were intraperitoneally immunized with the vaccines every 2 wk for a total of three times, and then subcutaneously challenged with Ehrlich ascites carcinoma (EAC) cells. Three weeks later, the mice were killed, and the tumors were surgically removed and weighed. Serum samples were collected from the mice, and antibody titers were determined by ELISA using an alkaline phosphate-conjugated detection antibody for total IgG. Antibody-dependent cell-mediated cytotoxicity was detected by the lactate dehydrogenase method using natural killer cells as effectors and antibody-labeled EAC cells as targets. Cytotoxic T lymphocyte activities were also detected by the lactate dehydrogenase method using lymphocytes as effectors and EAC cells as targets. RESULTS Vaccines were successfully synthesized and validated by analytical high performance liquid chromatography and electrospray mass spectrometry, including T7, T7-MG1, and T7-MG3. Rapid inductions of tumor necrosis factor-α and interleukin-12 in bone marrow dendritic cells and interferon γ and interleukin-12 in lymphocytes occurred in vitro after T7, T7-MG1, and T7-MG3 treatment. Immunization with T7-MG3 reduced the EAC tumor burden in BALB/c mice to 62.64% ± 5.55% compared with PBS control (P < 0.01). Six or nine weeks after the first immunization, the monoclonal gastric cancer 7 antigen antibody increased significantly in the T7-MG3 group compared with the PBS control (P < 0.01). As for antibody-dependent cell-mediated cytotoxicity, antisera obtained by immunization with T7-MG3 were able to markedly enhance cell lysis compared to PBS control (31.58% ± 2.94% vs 18.02% ± 2.26%; P < 0.01). As for cytotoxic T lymphocytes, T7-MG3 exhibited obviously greater cytotoxicity compared with PBS control (40.92% ± 4.38% vs 16.29% ± 1.90%; P < 0.01). CONCLUSION A successful method is confirmed for the design of gastric cancer vaccines by chemical conjugation of T7 and multi-repeat-epitope of monoclonal gastric cancer 7 antigen.

Synergistic apoptosis-inducing effect of aspirin and isosorbide mononitrate on human colon cancer cells

Aspirin and isosorbide mononitrate (ISMN) are two commonly used drugs, which are clinically applied for the treatment of inflammatory and cardiovascular diseases, respectively. Recently, aspirin has attracted interest due to its potential application for the treatment of cancer, particularly colon cancer. NO-aspirin, an aspirin derivative containing a covalently bound NO-donating moiety, has been proven to be an effective anti‑tumor agent with apoptosis-inducing ability. In the present study, ISMN was used as an NO donor and its synergic effect with aspirin was assessed in human colon cancer cells. In vitro, an MTT assay demonstrated that ISMN had a synergistic effect on the growth inhibitory effects of aspirin on HCT116 and SW620 colon cancer cells, while the growth of EA.hy926 normal endothelial cells was unaffected. This synergistic anti‑tumor effect was further validated in vivo using nude mouse HCT116 cell xenograft model. Observation of nuclear morphology, Annexin V-fluorescein isothiocyanate/propidium iodide double staining and a caspase-3 activity assay suggested that the combination of the two drugs induced apoptosis in HCT116 cells. Furthermore, the molecular mechanisms of the apoptotic effect of the drugs was assessed using an NO release assay, reverse transcription quantitative polymerase chain reaction analysis, western blot analysis and a luciferase reporter assay. It was certified that the increase in the amount of NO release, the decrease in the luciferase promoter activity and the expression of cyclin D1 and c-myc in HCT116 cells were affected by aspirin and ISMN in a synergistic manner. In conclusion, the present study was the first, to the best of our knowledge, to report on the synergistic apoptosis-inducing effects of aspirin and ISMN in human colon cancer cells, which were mediated via Wnt and NO signaling pathways. The results of the present study will facilitate the development of future therapeutic strategies.

Antitumor activity of a novel small molecule TLR7 agonist via immune response induction and tumor microenvironment modulation

Immunotherapy is emerging as a powerful and active tumor-specific approach against cancer via triggering the immune system. Toll-like receptors (TLRs) are fundamental elements of the immune system, which facilitate our understanding of the innate and adaptive immune pathways. TLR agonists used as single agents can effectively eradicate tumors due to their potent stimulation of innate and adaptive immunity. We examined the effects of a novel adenine type of TLR7 agonists on both innate and adaptive immune activation in vitro and in vivo. We established the local and distant tumor‑bearing mice derived from murine mammary carcinoma cell line (4T1) to model metastatic disease. Our data demonstrated that SZU101 was able to stimulate innate immune cells to release cytokines at the very high level compared with LPS at the same or lower concentration. Locally intratumoral SZU101 injection can elicit a systemic antitumor effect on murine breast tumor model. SZU101 affected the frequency of intratumoral immune cell infiltration, including the percentage of CD4+ and CD8+ increase, and the ratio of Tregs decrease. Our data reveal that the antitumor effect of SZU101 is associated with multiple mechanisms, inducing tumor‑specific immune response, activation of innate immune cells and modulation of the tumor microenvironment.

Toll‐Like Receptor 7 Inactive Ligands Enhanced Cytokine Induction by Conjugation to Weak Antigens

Toll‐like receptors (TLRs) 7/8 are key targets in the design and development of small‐molecule drugs serving as anticancer/antiviral agents and vaccine adjuvants. Clinical trials of imiquimod were discontinued owing to its serious adverse side effects. Herein we report the synthesis and biological evaluation of a series of 8‐hydroxy‐2‐(2‐methoxyethoxy)adenine derivatives that cannot induce cytokine production and that lack activity toward TLR 7/8. Their ability to triggering remarkable levels of cytokine production were revealed upon their conjugation with antigens that have weak immunogenicity. This discovery demonstrated that TLR 7 can be activated by coupling an antigen to the terminal carboxyl group at N9 of the inactive ligand adenine analogues. These inactive analogues may be well suited as new adjuvants with superior activity after conjugation, effectively decreasing the side effects caused by conventional adjuvants.

Immunomodulatory and Antitumor Effects of a Novel TLR7 Agonist Combined with Lapatinib.

As new treatment approaches, both immunotherapy and targeted treatments have been used in the clinical treatment of cancers. These therapies are different from traditional surgery, chemotherapy and radiotherapy. Use of a combination of immunotherapy and targeted treatments may improve tumor clearance. We investigated the feasibility of combining tyrosine kinase inhibitors (TKIs, targeted drugs) and SZU-101 (a novel TLR7 agonist synthesized by our laboratory). Thirteen different TKIs were combined with or without SZU-101 and studied to determine their effects on immunocytes. On the basis of the distinctive results, lapatinib and sunitinib were selected for further tumor-inhibition investigation and determination of the underlying mechanism. Interestingly, we found lapatinib to work better with SZU-101, enhancing tumor clearance in vivo, without affecting the TLR7-NF-κB pathway activated by the TLR7 agonist in mouse spleen lymphocytes and bone marrow dendritic cells (BMDCs).