DEVELOPMENT OF A TOXIC FUSION PROTEIN FOR BRAIN TUMORS

恶性脑肿瘤是最危及生命的癌症形式之一，胶质母细胞瘤是最具侵略性的脑癌形式。它占所有原发性脑肿瘤的约35%。美国每年大约有17，500例新的脑肿瘤病例。今天的标准疗法包括手术、放疗和化疗，对患者的生存影响相对较小。白细胞介素-4融合假单胞菌外毒素（IL-4PE）产生的靶向毒性蛋白可能为复发性胶质母细胞瘤提供新的治疗方法。PE是一种非常有效的毒素，当内化时，毒素会导致细胞死亡。恶性脑肿瘤表达丰富的IL-4受体，其以非常高的亲和力结合IL-4。因此，IL-4与PE的融合将提供通过内吞作用将外毒素递送到肿瘤细胞中的有效手段。Neurocrine已从FDA获得IL- 4PE的许可，并打算将这种融合蛋白开发成商业产品。为此，我们将首先构建一个表达载体，它可以在高产量表达蛋白。其次，我们将设计方法学来纯化和化学表征表达的蛋白质。第三，我们将建立一个体外细胞培养试验，以测量纯化的IL-4 PE的效力，最后，我们将制备放射性标记的IL-4 PE用于药代动力学研究，并开发一种双位点定向ELISA来测量动物和人体组织中的完整蛋白质。这些信息将有助于我们将IL-4PE推进到II期测试。拟议的商业应用：目前治疗脑肿瘤的疗法包括手术、放疗和化疗。然而，这些患者的预后仍然很差。将白细胞介素4与假单胞菌外毒素（IL-4PE）融合，开发出一种靶向毒素，可能为脑肿瘤患者提供有效的治疗。本申请中提出的研究将进一步发展这种新型治疗方法。

Malignant brain tumors are one of the most life-threatening forms of cancer and glioblastoma is the most aggressive form of brain cancer. It constitutes approximately 35% of all primary brain tumors. There are approximately 17,500 new cases of brain tumors in the U.S. annually. Today's standard therapies consisting of surgery, radiation and chemotherapy have a relatively small effect on the survival of the patients. A targeted toxic protein derived from fusion of interleukin-4 to Pseudomonas exotoxin (IL-4PE) may offer a new treatment approach for recurrent glioblastoma. PE is a very potent toxin and, when internalized, the toxin will cause cell death. Malignant brain tumors express abundant IL-4 receptors, which bind IL-4 with very high affinity. Thus, fusion of IL-4 with PE will provide an efficient means of delivering the exotoxin through endocytosis into the tumor cells. Neurocrine has licensed the IL- 4PE from the FDA and intends to develop this fusion protein into a commercial product. To do so we will first construct an expression vector, which can express the protein in high yield. Second, we will design methodology to purify and chemically characterize the expressed protein. Third, we will set up an in vitro cell culture assay to measure the potency of the purified IL-4PE and lastly we will prepare radiolabeled IL-4PE for pharmacokinetic studies and develop a two-site directed ELISA to measure the intact protein in animal and human tissues. This information will be helpful in our advancement of IL-4PE into Phase II testing. PROPOSED COMMERCIAL APPLICATIONS: The current therapy for treating brain tumors includes surgery, radiation therapy and chemotherapy. The prognosis for these patients however remains poor. The development of a targeted toxin derived from the fusion of IL-4 to Pseudomonas exotoxin (IL-4PE) may provide an effective treatment for those patients with brain tumors. The research proposed in this application will further the development of this novel therapeutic.