title: Conjugation of toll-like receptor-7 agonist to gastric cancer antigen MG7-Ag exerts antitumor effects.
abstract: OBJECTIVE
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Xiao-Dong Wang, Ning-Ning Gao, Yu-Wen Diao, Yu Liu, Dong Gao, Wang Li, Yan-Yan Wan, Jing-Jing Zhong, Guang-Yi Jin, School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, China.
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Wang
authors.affiliation: Xiao-Dong Wang, Ning-Ning Gao, Yu-Wen Diao, Yu Liu, Dong Gao, Wang Li, Yan-Yan Wan, Jing-Jing Zhong, Guang-Yi Jin, School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, China.
authors.forename: Ning-Ning
authors.initials: NN
authors.lastname: Gao
authors.affiliation: Xiao-Dong Wang, Ning-Ning Gao, Yu-Wen Diao, Yu Liu, Dong Gao, Wang Li, Yan-Yan Wan, Jing-Jing Zhong, Guang-Yi Jin, School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, China.
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Diao
authors.affiliation: Xiao-Dong Wang, Ning-Ning Gao, Yu-Wen Diao, Yu Liu, Dong Gao, Wang Li, Yan-Yan Wan, Jing-Jing Zhong, Guang-Yi Jin, School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Xiao-Dong Wang, Ning-Ning Gao, Yu-Wen Diao, Yu Liu, Dong Gao, Wang Li, Yan-Yan Wan, Jing-Jing Zhong, Guang-Yi Jin, School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Gao
authors.affiliation: Xiao-Dong Wang, Ning-Ning Gao, Yu-Wen Diao, Yu Liu, Dong Gao, Wang Li, Yan-Yan Wan, Jing-Jing Zhong, Guang-Yi Jin, School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, China.
authors.forename: Wang
authors.initials: W
authors.lastname: Li
authors.affiliation: Xiao-Dong Wang, Ning-Ning Gao, Yu-Wen Diao, Yu Liu, Dong Gao, Wang Li, Yan-Yan Wan, Jing-Jing Zhong, Guang-Yi Jin, School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, China.
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Wan
authors.affiliation: Xiao-Dong Wang, Ning-Ning Gao, Yu-Wen Diao, Yu Liu, Dong Gao, Wang Li, Yan-Yan Wan, Jing-Jing Zhong, Guang-Yi Jin, School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, China.
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Zhong
authors.affiliation: Xiao-Dong Wang, Ning-Ning Gao, Yu-Wen Diao, Yu Liu, Dong Gao, Wang Li, Yan-Yan Wan, Jing-Jing Zhong, Guang-Yi Jin, School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, China.
authors.forename: Guang-Yi
authors.initials: GY
authors.lastname: Jin
mesh_terms: D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D000951:Antigens, Neoplasm / Q000276:immunology*; D019496:Cancer Vaccines / Q000008:administration & dosage / Q000138:chemical synthesis / Q000276:immunology / Q000494:pharmacology*; D002286:Carcinoma, Ehrlich Tumor / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D000939:Epitopes; D005260:Female; D007115:Immunization Schedule; D018796:Immunoconjugates / Q000008:administration & dosage / Q000494:pharmacology*; D007274:Injections, Intraperitoneal; D016246:Lymphocytes, Tumor-Infiltrating / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000819:agonists* / Q000276:immunology / Q000378:metabolism; D008807:Mice, Inbred BALB C; D015398:Signal Transduction / Q000187:drug effects; D018089:Superantigens; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D013997:Time Factors; D051199:Toll-Like Receptor 7 / Q000819:agonists* / Q000276:immunology / Q000378:metabolism; D047368:Tumor Burden; D019139:Tumor Escape / Q000187:drug effects*
chemical_list: D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D016207:Cytokines; D000939:Epitopes; D018796:Immunoconjugates; D008562:Membrane Glycoproteins; D018089:Superantigens; C477038:Tlr7 protein, mouse; D051199:Toll-Like Receptor 7
keywords: Gastric cancer; Immunotherapy; Monoclonal gastric cancer 7 antigen; Toll-like receptor-7; Vaccine