Fully Synthetic Vaccines Consisting of Tumor-Associated MUC1 Glycopeptides and a Lipopeptide Ligand of the Toll-like Receptor 2
Fully Synthetic Vaccines Consisting of Tumor-Associated MUC1 Glycopeptides and a Lipopeptide Ligand of the Toll-like Receptor 2
复制标题
DOI：
10.1002/anie.201000462
复制
发表时间：
2010-01-01
影响因子：
16.6
通讯作者：
Kunz, Horst
中科院分区：
文献类型：
--
作者：
Kaiser, Anton;Gaidzik, Nikola;Kunz, Horst
上皮性肿瘤细胞上的粘蛋白糖蛋白结构与健康细胞上的粘蛋白结构有很大的不同。[1]然而，这些肿瘤相关糖蛋白的免疫原性太低，不能覆盖免疫系统的内源性耐受。因此，它们不能直接用作抗肿瘤疫苗。最近，有研究表明，来自肿瘤相关粘蛋白MUC1串联重复区域的糖肽与来自卵黄蛋白的T细胞表位肽结合可提供完全合成的疫苗，在转基因小鼠中诱导出强大的、高度特异的免疫反应。[2]以与破伤风类毒素结合的肿瘤相关MUC1糖肽为载体蛋白的疫苗免疫野生型Balb/c小鼠，可诱导更强、高特异性的免疫反应。当然，这种MUC1糖肽/破伤风类毒素疫苗也能诱导针对破伤风类毒素的免疫反应。为了抑制抗载体免疫反应的产生，例如在加强免疫中，必须开发一种替代形式的合成疫苗，其中与肿瘤相关的MUC1糖肽共价结合到一般的免疫刺激结构上。Toll样受体配体，例如Bessler，Jung等[4]描述的Pam3CysSer(Lys)4这样的三棕榈酰-S-甘油半胱氨酸肽就是这样的免疫刺激结构。最近，Boons等人[5]报道了由含有单糖TN抗原侧链的MUC1串联重复单元的十肽和前述TLR2激动剂直接偶联到脊髓灰质炎病毒的T细胞表位的疫苗。这些构建物在小鼠体内诱导了选择性免疫反应。在这些疫苗的合成过程中，N-乙酰半乳糖胺部分的O-脱乙酰基是通过与联氨在甲醇中的酯交换反应实现的。[7]这种方法不适用于含神经氨酸的糖肽的合成。为了利用Pam3Cys Toll样受体配体在补充破伤风类毒素结合物的合成MUC1糖肽疫苗中的免疫刺激作用，我们开发了一种片段缩合方法，将Pam3CSKKK脂肽连接到肿瘤相关的MUC1糖肽上，得到完全合成的疫苗A。为了最大限度地减少脂肽及其基本侧链对MUC1糖肽抗原构象的影响，在TLR2配体和B细胞表位之间放置了一个寡甘醇间隔物。只要脂肽的活性羧基上只含有酸性保护基团，糖肽的糖基部分已经被脱保护，最终的酸解脱保护应该不会影响棕榈酸酯，应该可以提供一种纯的全合成疫苗。用2-苯基-2-三甲基硅酸乙酯(PMTSEL)锚合成了N-末端和侧链保护的脂肽。[9]在中性条件下，该锚分子可在二氯甲烷中用三水四丁基氟化铵裂解。Fmoc-Lys(Boc)-OH与4-(2-羟基-1-三甲基硅乙基)苯氧乙酸烯丙基酯[9，11](1)按Steglich和Neise[10]的方法反应得到锚酯分子2(方案1)。用催化量的四(三苯基膦)钯(0)和N-甲基苯胺[12]作为烯丙基清除剂选择性地裂解烯丙基酯2。将得到的锚链羧酸3与氨基官能化的Tentagel[13]树脂用TbTU/HOBT偶联，得到树脂4-…
Mucin glycoprotein structures on epithelial tumor cells are characteristically different from the mucin structures on healthy cells.[1] However, the immunogenicity of these tumor-associated glycoproteins is too low to overwrite the endogenous tolerance of the immune system. Therefore, they can not be used directly as antitumor vaccines. Recently, it was demonstrated that glycopeptides from the tandem repeat region of tumor-associated mucin MUC1 conjugated to a T-cell epitope peptide from ovalbulmin furnish fully synthetic vaccines which elicit a strong, highly specific immune response in transgenic mice.[2] An even stronger and highly specific immune response was induced by immunization of wild-type balb/c mice with a vaccine containing the tumorassociated MUC1 glycopeptide bound to tetanus toxoid as the carrier protein.[3] This type of vaccines has the advantage of being applicable to humans. Of course, such MUC1 glycopeptide/tetanus toxoid vaccines also elicit immune reactions against tetanus toxoid. To suppress the generation of an anticarrier immune reaction, for example in booster immunizations, an alternative form of a synthetic vaccine must be developed in which the tumor-associated MUC1 glycopeptide is covalently bound to a general immunostimulating structure. Toll-like receptor ligands, for example tripalmitoyl-S-glycerylcysteine peptides like Pam3CysSer (Lys) 4 described by Bessler, Jung etal.,[4] represent such immunostimulating structures. Recently, Boons et al.[5] reported vaccines consisting of a glycoundecapeptide of the tandem repeat unit of MUC1 containing the monosaccharide TN-antigen side chain directly coupled to a T-cell epitope from polio virus [6] and the aforementioned TLR2 agonist. These constructs induced selective immune reactions in mice. During the synthesis of these vaccines the O-deacetylation of the N-acetylgalactosamine part was achieved by transesterification with hydrazine in methanol.[7] This procedure is not applicable to the synthesis of glycopeptides bearing neuraminic acid. To benefit from the immunostimulating effects of Pam3Cys Toll-like receptor ligands in synthetic MUC1 glycopeptide vaccines supplementing the tetanus toxoid conjugates, we developed a fragment condensation to attach the Pam3CSKKKK lipopeptide to tumor-associated MUC1 glycopeptides to give fully synthetic vaccines A.To minimize the influence of the lipopeptide and its basic side chains on the conformation of the MUC1 glycopeptide antigen, an oligoethylene glycol spacer was placed between the TLR2 ligand and the B-cell epitope. Provided the activated carboxylic group of the lipopetide bears only acidlabile protecting groups and the saccharide part of the glycopeptide already is deprotected, the final acidolytic deprotection should not affect the palmitic esters and should afford a pure fully synthetic vaccine. The N-terminally and side-chain-protected lipopeptide was synthesized on a resin functionalized with the 2-phenyl-2-trimethylsilylethylester (PMTSEL) anchor.[9] This anchor molecule is cleavable under neutral conditions by use of tetrabutylammonium fluoride trihydrate in dichloromethane. Fmoc-Lys (Boc)-OH was treated with 4-(2-hydroxy-1-trimethylsilylethyl) phenoxyacetic acid allyl ester [9, 11](1) according to the procedure reported by Steglich and Neises [10] to give the anchor ester molecule 2 (Scheme 1). The allyl ester 2 was cleaved selectively using catalytic amounts of tetrakis (triphenylphosphine) palladium (0) and N-methylaniline [12] as the allyl scavenger. The obtained anchor carboxylic acid 3 was coupled to amino-functionalized Tentagel [13] resin using TBTU/HOBt to yield the resin 4 …