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

项目总结/摘要 尽管在筛查和治疗方面取得了进展，但乳腺癌和肺癌的死亡率仍然很高， 美国在过去的20年里。人们普遍认为，早期发现对改善这两种疾病的结果至关重要。 疾病两者也都依赖于成像进行筛查，但假阳性和假阴性检测是不可能的。 与不必要的活检、漏诊和成本有关。迫切需要生物化学 提高成像技术性能的标记物。我们的实验室已经成功地 通过利用患者自身产生抗肿瘤抗体的能力来鉴定有用的癌症生物标志物， 相关抗原（TAA），称为肿瘤相关自身抗体（TAAb）。在EDRN的支持下， 我们开发了高通量可编程蛋白质展示方法，用于快速检测和验证 乳腺癌和肺癌中的自身抗体生物标志物特征。我们的乳腺癌TAAb生物标志物已经被 获得许可并集成到Videssa ™ Breast中，现在可作为CLIA认证的检测。我们的三重否定 乳腺癌标志物已经在盲态2期多中心验证研究中得到验证。这些 证明了TAAb在癌症早期检测中的巨大效用。然而，大多数TAAb的敏感性是 中度，建议通过检查可以获得更高的灵敏度和特异性 针对癌症中异常修饰蛋白的TAAb。我们的核心假设是异常蛋白质 糖基化，乳腺癌和肺癌的标志，诱导糖蛋白特异性TAAb， 作为这些癌症的特异性血清生物标志物。糖基化的改变是高度免疫原性的， 是对癌症改变的糖蛋白的显著抗体应答的强有力的历史证据。但所有 目前的蛋白质（或多肽）展示工具允许有限的翻译后修饰或不允许翻译后修饰。这一历史 由于缺乏筛选方法， 测试免疫原性结构糖蛋白。我们介绍了一种高通量显示全长的工具， 修饰有癌症特异性O-聚糖结构的蛋白质。这将彻底改变筛选的机会 聚糖蛋白表位在其自然环境中。我们的团队在功能蛋白质组学方面拥有丰富的专业知识， 生物标志物开发、糖蛋白组学、医学肿瘤学和生物统计学。靶向蛋白质将包括： 相关单程膜蛋白的细胞外结构域，已知是O-糖基化的蛋白 并且在两种癌症和所有已知的粘蛋白中过度表达。它们将被翻译在原地使用人类 核糖体和伴侣蛋白，然后用Tn和STn O-GalNAc型聚糖系统地修饰 通过连续添加重组糖基转移酶和糖核苷酸，以反映 两种癌症遵循PRoBE设计原则，我们将用癌症患者筛选这些阵列， 对照血清。我们的研究将集中在癌症患者和非癌症受试者与积极的成像结果。 研究设计将包括I期发现（阵列/ELISA）和使用ELISA的II期验证。

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.