NEO ANTIGEN AND IMMUNOBIOLOGY OF MEDULLARY BREAST CANCER

本研究旨在鉴定和表征在乳腺髓样癌（MC）中引起浆细胞反应的新表达蛋白（新抗原）。我们的主要假设是，浆细胞和淋巴细胞的显著浸润反映了局部对肿瘤的免疫反应，这是MIC肿瘤在手术治疗后更有利的自然病程的原因。我们将应用分子克隆技术从浸润肿瘤的浆细胞中获得抗体，然后使用这些抗体来检索肿瘤中新表达引起反应的蛋白质。我们从浆细胞浸润的MC肿瘤中制备了组合噬菌体Fab文库。我们证明这些组织表达高度集中的IgG抗体库，支持我们对肿瘤中表达的新抗原的特异性免疫反应的前提。此外，肿瘤中显性的类别切换IgG同型和高度的CDR突变提示t依赖抗原，即蛋白质。研究还表明，新抗原既不是p53也不是Her2/neu，这是两种主要的乳腺癌相关抗原，这更有可能（但尚未证明）新抗原是一种以前未被描述的蛋白质或以前未被认为与肿瘤相关的蛋白质。新抗原可以是外源（即病毒）蛋白，也可以是肿瘤中存在的异常表达或突变的正常蛋白。这些重要的初步数据证明了继续进行克隆诱导抗原的努力是正确的。抗原将通过筛选肿瘤特异性表达文库与患者抗体克隆，同时追求平行的生化方法，以确保克隆的成功。然后研究和评估克隆和表达的新抗原在恶性增殖中的可能作用。

The proposed research seeks to identify and characterize newly expressed proteins (neo-antigens) that cause plasma cell reactions in medullary carcinomas (MC) of breast. It is our principal hypothesis that the marked infiltration of plasma cells and lymphoid elements reflects a local immune response against tumor that is responsible for the impression of a more favorable natural course of MIC tumors after surgery-only therapy. We will apply molecular cloning techniques to derive antibodies from tumor-infiltrating plasma cells, and then use these antibodies to retrieve the proteins whose new expression in the tumors caused the response. We prepared combinatorial phage Fab libraries from plasma cell-infiltrated MC tumors. We proved these tissues express highly focussed IgG antibody repertoires, supporting our premise of a specific immune reaction against a neo-antigen expressed in the tumor. Further, the dominant class-switched IgG isotype in the tumor and the high degree of CDR mutations suggest a T-dependent antigen, i.e., a protein. It was also shown that the neo-antigen is neither p53 nor Her2/neu, two dominant breast carcinoma-associated antigens, making it more likely (but not yet proving) that the neo-antigen is a previously undescribed protein or a protein not previously recognized to be tumor-associated. The neo-antigen could be a foreign (i.e., viral) protein or an aberrantly expressed or mutated normal protein that is present in the tumor. These important preliminary data justify the pursuit of the remaining effort to clone the eliciting antigen. Antigen will be cloned by screening of tumor-specific expression libraries with patient antibodies, while pursuing a parallel biochemical approach to ensure success in the cloning. The cloned and expressed neoantigen(s) is(are) then studied and assessed for possible roles in the malignant proliferation.