Novel approaches to study immune responses to post translational modifications for cancer detection

项目摘要/摘要 尽管在筛查和治疗方面取得了进展，乳腺癌和肺癌的死亡率在#年仍居高不下。 在过去的20年里，美国。人们普遍认为，早期发现对改善这两种疾病的预后至关重要。 疾病。两者也都依靠成像进行筛查，但假阳性和假阴性检测是 与不必要的活组织检查、漏诊和费用有关。迫切需要生物化学 提高成像技术性能的标记。我们的实验室已经成功地在 通过利用患者自身产生抗肿瘤抗体的能力来识别有用的癌症生物标记物- 相关抗原(TAA)，称为肿瘤相关自身抗体(TAAb)。有了先前的EDRN支持， 我们开发了高通量可编程蛋白质显示方法，用于快速检测和验证 乳腺癌和肺癌中的自身抗体生物标记物特征。我们的乳腺癌TAAb生物标记物 获得许可并集成到Videssa™乳房中，现在可以作为CLIA认证的测试使用。我们的三重否定 乳腺癌标记物已在2期盲法多中心验证研究中得到验证。这些 展示了TAAb在癌症早期检测中的巨大作用。然而，大多数TAAbs的敏感性是 中度，而且有建议说，通过检查可以获得更高的敏感性和特异性 TAAb针对癌症中异常修饰的蛋白质。我们的中心假设是异常蛋白质 糖基化是乳腺癌和肺癌的标志，可诱导可测量的糖蛋白特异性TAAb 作为这些癌症的特异性血清生物标记物。糖基化的改变是高度免疫原性的，而且 是对癌症改变的糖蛋白产生显著抗体反应的强有力的历史证据。然而，所有的 目前的蛋白质(或多肽)展示工具允许有限的或没有翻译后修饰。这是历史上的 路障阻碍了这些生物标志物的识别，因为缺乏筛选方法 检测免疫原性结构糖蛋白。我们介绍了一种用于高通量显示全长图像的工具 带有癌症特有的O-糖链结构的蛋白质。这将彻底改变放映的机会 在其自然背景下的糖蛋白表位。我们的团队拥有强大的功能蛋白质组学专业知识， 生物标记物开发、糖蛋白组学、医学肿瘤学和生物统计学。目标蛋白质将包括： 相关单程膜蛋白的胞外结构域，已知是O-糖基化的蛋白质 并在两种癌症和所有已知的粘蛋白中过度表达。它们将使用人类在现场进行翻译 核糖体和伴侣蛋白，然后系统地用TN和STN O-GalNAc-型糖链修饰 通过连续添加重组糖基转移酶和糖核苷酸来反映在 两种癌症。本着探头设计的原则，我们将对这些阵列进行癌症患者和 对照血清。我们的研究将集中在癌症患者和影像呈阳性的非癌症受试者身上。 研究设计将包括第一阶段发现(ARRANSY/ELISA)和使用ELISA法进行第二阶段验证。

Project Summary/Abstract Despite advances in screening and treatment, mortalities from breast and lung cancers have remained high in the US over the last 20 years. It is widely accepted that early detection is critical to improving outcomes in both diseases. Both also rely on imaging for screening, but false positive and false negative detection are associated with unnecessary biopsies, missed diagnoses, and costs. There is an urgent need for biochemical markers that improve the performance of imaging technologies. Our laboratories have been successful at identifying useful cancer biomarkers by exploiting patients’ own ability to produce antibodies against tumor- associated antigens (TAA), referred to as tumor-associated autoantibodies (TAAb). With prior EDRN support, we developed high-throughput programmable protein display methods for the rapid detection and validation of autoantibody biomarker signatures in breast and lung cancers. Our breast cancer TAAb biomarkers have been licensed and integrated into Videssa™ Breast that is now available as CLIA-certified test. Our triple negative breast cancer markers have been validated in blinded phase 2 multicenter validation studies. These demonstrate the great utility of TAAb in cancer early detection. However, the sensitivities of most TAAbs is moderate and there is a suggestion that greater sensitivity and specificity could be obtained by examining TAAb directed at aberrantly modified proteins in cancers. Our central hypothesis is that aberrant protein glycosylation, a hallmark of breast and lung cancers, induces glycoprotein-specific TAAb that can be measured as specific serum biomarkers of these cancers. Alterations in glycosylation are highly immunogenic, and there is strong historical evidence for significant antibody responses to cancer-altered glycoproteins. However, all current protein (or polypeptide) display tools allow limited or no post-translational modification. This historical roadblock has prevented the identification of these biomarkers because of the lack of screening methods that test immunogenic structural glycoproteins. We introduce a tool for the high-throughput display of full-length proteins decorated with cancer-specific O-glycan structures. This will revolutionize the opportunity to screen glycan-protein epitopes in their natural context. Our team comprises strong expertise in functional proteomics, biomarker development, glycoproteomics, medical oncology and biostatistics. Targeted proteins will include: the extra-cellular domains of relevant single pass membrane proteins, proteins known to be O-glycosylated and overexpressed in the two cancers, and all known mucins. They will be translated in situ using human ribosomes and chaperone proteins and then systematically decorated with Tn and STn O-GalNAc-type glycans by consecutive addition of recombinant glycosyltransferases and sugar nucleotides to mirror what occurs in the two cancers. Adhering to the principles of PRoBE design, we will screen these arrays with cancer patient and control sera. Our study will focus on cancer patients and non-cancer subjects with positive imaging findings. Study design will include Phase I discovery (arrays/ELISA) and Phase II validation using ELISA.