2013 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, 理学研究科, 教授
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| Co-Investigator(Kenkyū-buntansha) |
CHANG Tsung-che 大阪大学, 理学研究科, 外国人特別研究員
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| Keywords | Cancer vaccine / Sialvlation / Indium (III) trifluoromethanesulfonate |
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| Research Abstract |
Cell surface display of aberrant levels and types carbohydrates domains of glycoprotein and glycolipids differentiate cancer cells from normal cells for the elimination in the immune system. These tumor-associated carbohydrae antigens (TACAs) have made it possible to develop the anti-cancer vaccines. To improve the presentation of a TACA to relevant immune cells is to attach the antigen to a receptor ligand that can activate appropriate immune cells. TLR ligands, such as mono-phosphoryl lipid A (MPL) (TLR4 ligand), or bacterial lipopeptides (TLR2 ligand), were developed as an adjuvant for various virus vaccines, but there have appeared some issues on them. Our lab has newly developed adjuvants that control certain pathways of the immune system with the combination of some receptor ligands, which include Nodl, Nod2 or special-types of TLR4 ligands. I will thus combine these adjuvants with TACAs to form a cancer vaccine. The synthesized vaccine would show more efficient class switch to I
… More
gG antibodies against tumor-associated carbohydrate epitopes.
The sTn antigens are expressed in carcinoma-associated mucins. Mucins are highly Ο-glycosylated, high molecular weight glycoproteins expressed on endodermal epithelial cells. However, the sTn-based vaccine, (Theratope) for metastatic breast cancer, failed in phase III. Modification of the C5 N-substitutent of sialic acid in sialic-acid-containing antigens has been shown to enhance their immunogenicity. Therefore, I have synthesized a series of sTn derivative-KLH conjugates and found the N-propionyl sTn antigen was the most immunogenic among the sTn derivatives investigated. Hence, the N-modification sTn may serve as a viable TACA candidate.
First of all, the synthesis of Fmoc/tert-butyl ester protected Tn antigen threoninc building blocks bearing isopropylidene protection I was achieved according to the following progress. D-Galactose was transformed into 2-azidogalactosyl bromide 2 as a glycosyl donor in three steps. After glycosylation between compound 2 and acceptor 3. Finoc-thrconine-Ο-^1Bu according to modified Koenigs-Enorr methodology, theΟ-glycosyl amino acid 5 was transformed into fully protected Tn antigen conjugate 6 through reduction of the azido group and acetylation. After careful removal of the Ο-acetyl groups of 6, the 3- and 4-hydroxy groups were protected by using isopropylidene group to form compound 1 (overall yield 10%, 9 steps). Subsequently. The N-azido neuraminic acid donor, prepared from neuraminic acid, was coupled with compound 1 to create 2,6-linked sialoside 7 in good yield (94% brsm) and selectivity (α/β= 92/8) by applying ICI and In (OTf)_3 system. After removing isopropylidene by acetic acid aqueous and forming acetylation from compound 7, the azido group of sialoside was reduced by Cu/Zn powder in quantitive yield, followed by acetylation or propylation in excellent yield to afford N-acetyl or propionyl sTn antigen, respectively. Then, N-acetyl sTn was removed tert-butyl group in acidic condition to afford suitable building unit 8 for trimeric N-acetyl sTn assembly. The building unit 8 was coupled with azido-triethylene glycol amine by amide bond formation to form compound 9. Glycopeptide 9 was then carried through the sequence of deprotections and coupling with 8 using the HOAt-HATU coupling method to form trimeric N-acetyl sTn. The designed clustered epitopes might posses a more accurate representation or mimic the highly glycosylated tumor cell surface, which in turn could enhance immunogenicity. Until now, I have synthesized the trimeric N-acetyl sTn antigen and N-propionyl sTn antigen. Less
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Until now, the progress of research meets the performance of expectation.
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| Strategy for Future Research Activity |
After synthesizing the trimeric N-acetyl and proionyl sTn antigen as the TACA candidates, I will check the following combination of our adjuvant library ; i) PGN fragment (Nodl or Nod2 ligands) that induce moderate immune responses, ii) TLR2 ligands such as bacterial lipopeptides of which activity is controlled by peptide sequence, iii) library of lipid A and analogs. Based on the results, the appliacnt will synthesize newly designed conjugated adjuvants. With respect to PGN fragment, γ-D-Glu-meso-diaminopimelic acid (iE-DAP) as Nod I ligand and muramyl dipeptide (MDP) as Nod2 ligand would be synthesized. The synthesis of lipopeptides which contained the S-[2,3-bis (palmitoyloxy) propyl] cysteine moiety (Pam2Cys) would be achieved with a combination solution and solid-phase method.
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