Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes.
Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes.
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DOI：
10.1021/bc300422a
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发表时间：
2013-03-20
影响因子：
4.7
通讯作者：
Sucheck, Steven J.
中科院分区：
文献类型：
--
作者：
Sarkar, Sourav;Salyer, Alex C. D.;Wall, Katherine A.;Sucheck, Steven J.
与肿瘤相关碳水化合物抗原(TACA)结合的MUC1可变数目串联重复序列(VNTRs)在人源化MUC1转基因小鼠中被证明打破了自身耐受性。因此，我们假设MUC1VNTRTACA结合物可以成功地制成脂质体为基础的抗癌疫苗。疫苗的免疫原性应该通过将表面显示的L-鼠李糖(Rha)表位整合到脂质体上来进一步增强，以利用天然的抗体依赖的抗原摄取机制。为了验证我们的假设，我们用SPPS合成了一个含有GalNAc-O-Thr(TN)TACA的20个氨基酸的MUC1糖肽，并将其连接到一个功能化的Toll样受体配体(TLRL)上。以四甘醇(TEG)为连接物，制备了L-Rha-胆固醇偶联物。以TLRL-MUC1-TN结合物、Rha-TEG-胆固醇和1，2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)为原料，在总脂浓度为30 mM的条件下，采用挤出法制备了脂质体抗癌疫苗。通过扫描电子显微镜和差示扫描量热仪对脂质体的稳定性、均一性和粒径进行了评价。制备的脂质体与抗Rha抗体和鼠抗人MUC1抗体均呈阳性结合。将以明矾为佐剂的鼠李糖-Ficoll(Rha-Ficoll)结合物免疫雌性BALB/c小鼠，免疫小鼠产生适当浓度的抗Rha抗体。在用Rha-Ficoll结合物免疫的小鼠中，抗Rha抗体效价是未免疫对照组的25倍。然后用TLRL-MUC1-TN脂质体疫苗免疫小鼠，无论是用还是不用表面显示Rha表位的脂质体疫苗。从最初接种Rha-Ficoll和后来接种显示Rha-Ficoll的TLRL-MUC1-TN脂质体的小鼠中收集的血清显示，与没有接种Rha-Ficoll的小鼠相比，抗MUC1-TN和抗TN抗体效价都增加了8倍。在Rha-Ficoll免疫小鼠分离的抗体存在下，BALB/c小鼠的T细胞经MUC1-TN多肽免疫后对Rha脂质体疫苗的增殖作用高于非免疫小鼠，这支持了所提出的抗原提呈效应。免疫小鼠血清中的抗MUC1-TN抗体可识别人白血病U266细胞上的MUC1。由于这种疫苗使用不同的鼠李糖和抗原表位成分，因此疫苗可以通过改变肽来轻松地针对不同的抗原或表位，而不必改变其他成分。
MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anti-cancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface displayed L-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis we synthesized a 20-amino acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An L-Rha-cholesterol conjugate was prepared using tetraethylene glycol (TEG) as a linker. The liposome-based anti-cancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the non-immunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.
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DOI：
10.1016/j.bbrc.2005.02.092
发表时间：
2005-04-22
影响因子：
3.1
作者：
Bardonnet, PL;Faivre, V;Falson, F
通讯作者：
Falson, F
影响因子：
16.6
作者：
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通讯作者：
Kunz, H
DOI：
10.1073/pnas.1115166109
发表时间：
2012-01-03
影响因子：
11.1
作者：
Lakshminarayanan, Vani;Thompson, Pamela;Boons, Geert-Jan
通讯作者：
Boons, Geert-Jan
影响因子：
5.4
作者：
Abdel-Motal, Ussama;Wang, Shixia;Galili, Uri
通讯作者：
Galili, Uri
影响因子：
5.5
作者：
Foged, C;Arigita, C;Frokjaer, S
通讯作者：
Frokjaer, S