GRSA Proteins in the Diagnosis of Breast Cancer

描述(申请人提供)：本项目的目标是测试 改进乳腺癌诊断技术的假设可能是 由识别GRSA蛋白质的试剂产生。这些代理包括 单抗和工程单链可变区(ScFv)形式 这些抗体中。糖肽相关细胞表面抗原蛋白 是由加压素的表达产生的特定的肿瘤产物 (VP)基因。它们似乎都是由乳腺癌和导管引起的。 原位癌(DCIS)。另外，VP基因表达和GRSA蛋白 在正常乳腺组织和所有乳腺纤维囊性疾病中均不存在 包括不典型导管增生(ADH)。对这种区别的确认 应提供一种早期发现初发和复发的方法 疾病。初步研究表明，GRSA蛋白可能是一个靶标 用于免疫诊断和未来的治疗干预。因此，一个 资助期间研究的主要重点是生成和评估 为了这些目的，scFv GRSA从可用的抗体中定向分子。 具体目标是：(I)评估新的免疫组织化学和RT-PCR 以单克隆为特征的乳腺癌和DCIS的诊断程序 抗GRSA蛋白抗体或VPmRNA引物；(Ii)制造 代表我们的单抗的单链单链抗体和三联体 识别乳腺癌GRSA蛋白的抗体；(Iii)评价 基于其功能，这些单链抗体分子在体外可作为有用的临床制剂 提供档案中所有乳腺癌的阳性免疫染色 文库，并与培养中的癌细胞结合；以及；(Iv)鉴定这些单链抗体 基于分子在体内作为有用的临床制剂的能力 选择性靶向小鼠肿瘤移植瘤上的GRSA蛋白。研究将雇用 RT-PCR，连接和克隆；ABC免疫组织化学，Western分析；Flow 细胞术；放射免疫分析；组织放射分析；伽马相机闪烁成像。 这项研究的预期结果是开发简单、敏感、 区分DCIS的可靠且普遍可用的诊断方法 和乳腺癌组织活检中的非典型增生，这可以 通过放射免疫成像检测和定位患者的复发疾病。 这些方法可能适用于所有乳腺癌患者或 疑似乳腺癌。

DESCRIPTION (provided by applicant): The objective of this project is to test the hypothesis that improved diagnostic techniques for breast cancer can be generated with agents recognizing GRSA proteins. Such agents comprise monoclonal antibodies and engineered single-chain variable region (scFv) forms of these antibodies. GRSA (Glycopeptide-Related Cell Surface Antigen) proteins are specific tumor products that result from the expression of the vasopressin (VP) gene. They are produced by seemingly all breast cancer and all ductal carcinoma in Situ (DCIS). Alternatively, VP gene expression and GRSA proteins are absent from normal breast tissue and from all breast fibrocystic conditions including atypical ductal hyperplasia (ADH). A confirmation of this distinction should provide a method for the early detection of initial and recurrent disease. Preliminary studies indicate GRSA proteins could represent a target for immunodiagnosis and for future therapeutic interventions. Therefore a principal focus of research over the funding period is to generate and evaluate ScFv GRSA-directed molecules from available antibodies for these purposes. Specific Aims are directed at: (i) assessing new immunohistochemical and RT-PCR diagnostic procedures for breast cancer and DCIS that feature monoclonal antibodies against GRSA proteins or primers for VPmRNA; (ii) manufacturing single-chain scFv diabodies and triabodies representing our monoclonal antibodies that recognize GRSA proteins on breast cancer; (iii) appraising these scFv molecules as useful clinical agents in vitro based on their ability to provide positive immunostaining of all breast carcinoma in an archival library, and to bind to cancer cells in culture, and; (iv)appraising these scFv molecules as useful clinical agents in vivo based on their ability to selectively target GRSA proteins on mouse tumor xenografts. Studies will employ RT-PCR, ligation, and cloning; ABC immunohistochemistry, Western analysis; flow cytometry; RIA; radiometric analysis of tissues; and gamma-camera scintigraphy. Expected outcomes of this research are the development of simple, sensitive, reliable, and universally available diagnostic methods that distinguish DCIS and breast cancer from atypical hyperplasia in tissue biopsies, and that can detect and localize recurrent disease in patients through radioimmunoimaging. These methods could be applicable to all patients with breast cancer or suspected breast cancer.