A Novel Diagnostic Assay for Oncogenic Human Papillomaviruses

描述(由申请人提供)：Arbor Vita Corporation(AVC)基于对人类乳头瘤病毒(HPV)生物学的了解，开发了一种新的宫颈癌诊断方法。HPV感染是最常见的性传播疾病之一，仅在美国估计每年就有550万新感染病例。高危(致癌)HPV类型几乎与所有宫颈癌相关。巴氏涂片和液基细胞学筛查大大降低了宫颈癌的发病率，但巴氏试验的假阴性率和假阳性率都很高，需要训练有素的细胞学家的广泛基础设施来解释结果。一种更便宜、更可靠、更具预测价值的测试将会对临床产生巨大的好处。高危HPV类型的病毒编码的E6和E7蛋白被证明是细胞转化和癌症进展所必需的，而高危HPV类型的E6蛋白，而不是低风险HPV类型的E6蛋白，已知与细胞PDZ结构域结合。AVC扩展了这些研究，并证明了高危HPV和E6-PDZ结合之间的完美相关性。基于这些发现，我们开发了一种新的利用PDZ蛋白捕获的HPVE6诊断宫颈癌的方法。在我们的SBIR第一阶段，AVC开发了一种针对HPVE6的新型PDZ捕获夹心ELISA法，该方法可以检测75%以上的高危HPV类型，并在人类癌症样本中展示了其有效性。我们能够开始对细胞中的E6进行量化，并提高了灵敏度，以允许在比巴氏试验通常收集的样本少得多的样本中检测到E6。在第二阶段，我们建议完成基于PDZ的宫颈癌检测原型的开发，为临床试验做准备。具体地说，我们建议：1.扩大我们的抗体检测范围，将95%的已知高危HPV类型包括在内。2.优化E6蛋白检测的临床样品处理。3.优化PDZ/抗体夹心ELISA法用于临床实验室。4.扩大高危E6蛋白水平与临床细胞学分期的相关性研究。

DESCRIPTION (provided by applicant): Arbor Vita Corporation (AVC) has developed a novel cervical cancer diagnostic based on an understanding of the biology of human papillomavirus (HPV). HPV infection is one of the most common sexually transmitted diseases with an estimated 5.5 million new infections per year in the U.S. alone. High-risk (oncogenic) HPV types are correlated with virtually all cervical cancers. Pap smear and liquid based cytology screening has greatly reduced the incidence of cervical cancer, but the Pap test has both high false-negative and false-positive rates and requires an extensive infrastructure of trained cytologists to interpret the results. A cheaper test with greater reliability and predictive value would be of great clinical benefit. The virally-encoded E6 and E7 proteins of high-risk HPV types have been shown to be essential for cell transformation and cancer progression and E6 proteins from high-risk HPV types, but not low-risk HPV types, are known to bind cellular PDZ domains. AVC has extended those studies and demonstrated a perfect correlation between high-risk HPV and E6-PDZ binding. Based on these findings, we have developed a novel cervical cancer diagnostic assay of HPV E6 using PDZ protein capture. In our SBIR Phase I, AVC developed a novel PDZ capture sandwich ELISA test for HPV E6 that detects over 75% of high-risk HPV types and demonstrated its utility with human cancer samples. We were able to begin quantifying E6 from cells and improved sensitivity to allow E6 detection in a much smaller sample than typically collected in a Pap test. In Phase II, we propose to complete development of our prototype PDZ-based cervical cancer test in preparation for clinical trials. Specifically, we propose to: 1. Expand our antibody detection to include 95% of known high-risk HPV types. 2. Optimize clinical sample handling for E6 protein detection. 3. Optimize the PDZ/antibody sandwich ELISA for clinical laboratory implementation. 4. Extend the studies correlating high-risk E6 protein levels and clinical cytology staging.