分布于细胞膜的Mammaglobin-A免疫表位鉴定及其作为乳腺癌治疗分子靶标的实验研究

Mammaglobin A (Mam-A) is an breast organ specific molecular biomarker with a promising prospect for anticancer therapy. However, due to the the late discovery of distribution of the Mam-A in the cell membrane, unknown of the membrane related immune epitopes and the scarcity of corresponding monoclonal antibody, the progress of MGA targeting anticancer therapy has been seriously hindered. Our previous study successfully prepared theory covering Mam-A all immune epitope monoclonal antibody. The preliminary results showed some of specifically cell membrane targeting antibodies can recognize different Mam-A epitopes. In this project, we aim to prepare Mam-A ploypeptide fragments with adjacent segments overlapping method followed by antibody polypeptide reaction and polypeptide pre sealed the antibody /Mam-A reaction blockade, the identification Mam-A potential cell membrane epitope and its sequence. Subsequently, by targeting different epitopes of the monoclonal antibody, we will carry out Mam-A targeting anti cancer therapy, to elucidate the the effects and mechanisms of Mam-A targeting therapy. Finally, through conjugating the different epitope targeting monoclonal antibodies with photosensitizer and chemotherapy drugs, we will carriy out based on the Mam-A target of immunotherapy treatment research to clarify potential value of the molecular targets in the treatment of breast cancer.

乳腺球蛋白A(Mammaglobin-A,Mam-A)在乳腺癌中特异高表达，被公认为很有前景的乳腺癌治疗分子靶标。然而，由于Mam-A在细胞膜的分布发现较晚、对其免疫表位的未知及相应单抗的稀缺等问题，严重阻碍了本领域进展。申请人前期成功制备了理论上覆盖Mam-A所有免疫表位的数十株单抗，初步研究表明多个特异靶向细胞膜Mam-A区段的单抗识别表位存在不同。本项目拟在此基础上，以相邻片段部分重叠的方式制备Mam-A蛋白的系列多肽片段，通过抗体-多肽反应及多肽预封闭对抗体/Mam-A反应的阻断情况，鉴定Mam-A潜在细胞膜表位及其序列。之后，利用靶向不同表位的单抗，深入开展基于Mam-A为靶标的乳腺癌单抗治疗研究，阐明靶向不同表位单抗的治疗效应及其机制；同时，利用靶向不同表位的单抗偶联光敏剂以及化疗药物，开展基于Mam-A为靶标的免疫导向治疗研究，阐明该分子靶标用于乳腺癌治疗的潜在价值。

课题承担团队按照项目计划书要求完成了全部研究内容，并取得了系列重要研究成果，包括：成功筛选并鉴定了Mam-A分子7个潜在免疫表位，并分别获得了针对不同表位的单克隆抗体，本成果是目前对Mam-A分子免疫特征最全面的解析，为阐明以往基于Mam-A的不同研究中结论不一等问题提供了依据，同时为今后基于Mam-A的乳腺癌诊断、靶向治疗等研究中优化表位与抗体选择、提高研究成果的可靠性与可重复性提供了支撑；在以往报道的基础上，鉴定出两个分布于细胞膜的新表位，并分别通过体外实验明确了针对Mam-A不同表位直接进行抗体治疗均不具有明显抗乳腺癌意义；进一步，通过抗体偶联光敏剂及纳米药物，证明了靶向Mam-A能够显著提高抗乳腺癌效能，为Mam-A作为分子靶标的研究明确了可行方向。在本项目资助下，2项重要研究成果发表于本领域Top期刊（1篇IF>10,1篇IF>15），获得3项国家发明专利授权，其中1项关于Mam-A抗体的专利具有重要经济价值，已进一步用于乳腺癌诊断试剂的研发。