Specificity analysis of sera from breast cancer patients vaccinated with MUC1-KLH plus QS-21
Specificity analysis of sera from breast cancer patients vaccinated with MUC1-KLH plus QS-21
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DOI：
10.1038/sj.bjc.6690288
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发表时间：
1999-04-01
影响因子：
8.8
通讯作者：
Livingston, P
中科院分区：
文献类型：
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作者：
Adluri, S;Gilewski, T;Livingston, P
与正常组织相比，黏液蛋白MUC1在乳腺癌中以糖基化程度较低的形式表达，因此是癌症免疫治疗的潜在靶点。MUC1含有20个氨基酸（aa）肽（VTSAPDTRPAPGSTAPPAHG）的多个串联重复序列。APDTRPA表位特别具有免疫原性，因为它可以被多种小鼠单克隆抗体和一些来自未免疫的上皮癌患者的血清和细胞毒性t细胞识别。我们制备了一种n端含有半胱氨酸的30 aa肽(C)VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA，并将该半胱氨酸与keyhole帽状血青素（KLH）进行化学偶联，用该偶联物与免疫佐剂q -21免疫的6例乳腺癌患者均产生了高滴度（ELISA法）的针对30 aa MUC1肽的IgG和IgM抗体，但这些血清与MUC1阳性肿瘤细胞反应一般，或根本不反应。为了更好地理解这种特异性，我们制备了一系列较小的肽来确定这些免疫血清在抑制试验中识别的表位。只有在c端含有APDTRPA的肽能够完全抑制整个30aa肽的ELISA反应性。没有血清被APDTR、APDTRP、PDTRPA或任何其他不含完整APDTRPA表位的肽完全抑制。值得注意的是，所有6名患者的血清都能识别出相同的表位，并且仅被该表位完全抑制。这些血清的特异性(1)主要针对c端APDTRPA，并且在任何肿瘤粘蛋白的c端都不存在该表位，(2)n端APDTRPA丙氨酸通常埋在MUC1的二级结构中，这可能解释了这些高滴度血清对MUC1阳性肿瘤细胞的中度至弱反应性。
The mucin MUC1 is expressed on breast cancers in an underglycosylated form compared to normal tissues and is therefore a potential target for cancer immunotherapy. MUC1 contains multiple tandem repeats of the 20 amino acid (aa) peptide (VTSAPDTRPAPGSTAPPAHG). The APDTRPA epitope is particularly immunogenic since it is recognized by a variety of murine monoclonal antibodies and by some sera and cytotoxic T-cells from unimmunized patients with epithelial cancers. We have prepared a 30 aa peptide (C)VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA with cysteine at the N-terminal end, and used the cysteine for chemical conjugation to keyhole limpet haemocyanin (KLH), Six breast cancer patients immunized with this conjugate plus the immunological adjuvant QS-21 have all produced high titre (by ELISA) IgG and IgM antibodies against the 30 aa MUC1 peptide, but these sera reacted moderately, or not at all, with MUC1-positive tumour cells. To understand this specificity better, we prepared a series of smaller peptides to determine the epitopes recognized by these immune sera in inhibition assays. Only peptides containing APDTRPA at the C-terminal end were able to completely inhibit ELISA reactivity for the full 30 aa peptide. No sera were completely inhibited by APDTR, APDTRP, PDTRPA or any other peptides that did not contain the full APDTRPA epitope. Remarkably, sera from all six patients recognized this same epitope and were completely inhibited by only this epitope. The specificity of these sera (1) primarily for C-terminal APDTRPA, and the absence of this epitope at the C-terminal end of any tumour mucins, and (2) the N-terminal APDTRPA alanine, which is normally buried in the beta turn MUC1 assumes in its secondary structure may explain the moderate to weak reactivity of these high titer sera against MUC1-positive tumour cells.