Functional Targeting of the Tyrosine Kinase EphA5 in Radiation-resistant Lung Cancer

放射治疗（RT）是治疗非小细胞肺癌（NSCLC）的综合治疗方式。 然而，肺癌通常对RT和介导治疗抗性的分子机制是难治的， 肿瘤再增殖仍然不明确。我们已经确定受体酪氨酸激酶EphA 5是 在肺癌中高表达，更重要的是，其在患者中的表达与RT呈负相关， 成功和生存，从而表明其参与调节细胞对遗传毒性损伤的反应。 我们已经收集了多条证据来支持EphA 5介导的细胞凋亡的潜在机制。 辐射抗性：（i）EphA 5沉默的肺癌细胞显示出缺陷的G1/S细胞周期检查点， 不能解决DNA损伤，（ii）在辐射时，EphA 5在细胞核中被发现，在那里它与 与激活共济失调毛细血管扩张症突变（ATM）的DNA修复位点，（iii）我们证明了一个新的 针对EphA 5的单克隆抗体使肺癌细胞和人肺癌异种移植物对RT敏感， 显著延长荷瘤小鼠的存活。为了开始翻译我们的发现，我们有 开发了一种新的方法，该方法利用基于噬菌体的体外和体内筛选方法， 酵母抗体展示以选择和表征具有放射增敏特性的抗体，并且能够 识别体内的目标。在目标1中，我们将应用我们的组合分级方法来产生抗EphA 5 具有放射增敏特性并且能够在体内靶向表达EphA 5的肿瘤的抗体。目的2 将研究EphA 5在DNA损伤反应中的功能，以及如何预防它以达到治疗目的。目标3 将定义所选抗体的生物分布特性。毒理学研究和探索性研究 还将进行药效学/药代动力学研究。在目标4中，我们将测试靶向抑制是否 EphA 5与电离辐射的结合将改善治疗结果。

Radiation therapy (RT) is an integral therapeutic modality for treating non-small cell lung cancer (NSCLC). However, lung cancer is often refractory to RT and molecular mechanisms mediating treatment resistance and tumor repopulation remain poorly defined. We have determined that the receptor tyrosine kinase EphA5 is highly expressed in lung cancer and, more importantly, its expression in patients negatively correlates with RT success and survival, thus suggesting its involvement in the regulation of cellular responses to genotoxic insult. We have assembled multiple lines of evidence to support a potential mechanism underlying EphA5-mediated radioresistance: (i) EphA5-silenced lung cancer cells display a defective G1/S cell cycle checkpoint and are unable to resolve DNA damage, (ii) upon irradiation, EphA5 is found in the nucleus of cells where it interacts with activated ataxia-telangiectasia mutated (ATM) at sites of DNA repair and, (iii) we demonstrated that a new monoclonal antibody against EphA5 sensitizes lung cancer cells and human lung cancer xenografts to RT, and significantly prolongs survival of tumor-bearing mice. In order to initiate the translation of our findings, we have developed a new methodology that utilizes in vitro and in vivo screening approaches based on phage and yeast antibody display to select and characterize antibodies with radiosensitizing properties, and capable of recognizing targets in vivo. In Aim 1, we will apply our combined hierarchical approach to generate anti-EphA5 antibodies with radiosensitizing properties and capable of targeting EphA5-expressing tumors in vivo. Aim 2 will study functions of EphA5 in DNA damage response, and how to prevent it for therapeutic purposes. Aim 3 will define biodistribution properties of selected antibodies. Toxicology studies, and exploratory pharmacodynamics/pharmacokinetics will also be performed. In Aim 4, we will test whether targeted inhibition of EphA5 combined with ionizing radiation will improve treatment outcomes.