2014 Fiscal Year Annual Research Report
| Project/Area Number |
13F03329
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| Research Institution | Osaka University |
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Principal Investigator |
深瀬 浩一 大阪大学, 理学(系)研究科(研究院), 教授 (80192722)
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| Co-Investigator(Kenkyū-buntansha) |
CHANG Tsung-Che 大阪大学, 理学(系)研究科(研究院), 外国人特別研究員
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| Project Period (FY) |
2013-04-01 – 2016-03-31
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| Keywords | Tumor-associated antigen / Sialyl-Tn / Anticancer vaccine / Adjuvant / Innate immunity / Adaptive immunity |
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| Outline of Annual Research Achievements |
Last year, the trimeric N-acetyl sTn antigen and N-propionyl sTn antigen as epitope of B cell had been synthesized. However, the T cell epitope and adjuvant (Pam3CSK4) were necessary to be synthesized for constructing the full synthetic anticancer vaccine. Based on the standard protocol of solid phase peptide synthesis, the P2 fragment of Tetanus Toxoid as T cell epitope was synthesized. The N-terminal of the T cell epitope was alkyne group for copper-click conjugation. The lipopeptide containing the bromoacetyl group for nucleophilic substitution was also smoothly achieved by the solid phase synthesis in good yield. After the surveying many conditions of conjugation between trimeric sTn antigen and T cell epitope, the copper-click conjugation was achieved successfully in good yield to afford compound 1. Then, the above conjugated compound 1 was removed fmoc group smoothly for forming disulfide bond to afford compound 2 in high yield. Unfortunately, the epimerization happened during the fully deprotection of acetate group and ester group under basic condition. The fully deprotection of acetate group of trimeric sTn antigen was done smoothly in quantitative yield without epimerization happened to afford compound 3. The compound 3 was conjugated with T cell epitope through copper-click chemistry by oxygen-free solvent to get compound 4. The compound 4 was also removing the fmoc group to disclose thiol group to afford compound 5 without epimerization happened. Finally, the lipopeptide (Pam3CSK4) is being conjugated with compound 5 by forming thioether bond now.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Based on the predetermined schedule, the fully synthetic self-adjuvanting N-modified sTn Antigen as anticancer vaccine should be finished on the end of January in 2015. And then the immune assay will also be started to proceed on the end of March this year. However, the conjugation of B cell epitope (Trimeric sTn) and T cell epitope (P2 fragment) took lots of time to look for the optimal method to avoid the unwanted side reaction. Moreover, the time of synthesizing B cell epitope, T cell epitope, and adjuvant was time-consuming. The above reasons made the progress to be delayed around 3 months. Fortunately, the syntheses of methods in key steps were found now. The progress would be proceeded smoothly in next step.
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| Strategy for Future Research Activity |
After finishing the fully synthetic self-adjuvanting N-modified sTn Antigen as anticancer vaccine, the resulting fully synthetic vaccines will be used for the vaccination of mice, either in a liposome with Freund’s adjuvant or in aqueous PBS buffer. The induced antibodies will be evaluated their end-point titer by ELISA of anitsera. After that, the binding of the induced sera to tumor cells will also determined by flow cytometry experiment (FACS). Finally, the initiated the killing of the recognized tumor cells through activation of the complement-dependent cytotoxicity (CDC) of mice also be evaluated. We expect the the synthesized vaccine would show more efficient class switch to IgG antibodies against tumor-associated carbohydrate epitopes (TACAs) to override the natural tolerance of TACAs in immune response.
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