DEVELOPMENT OF A TOXIC FUSION PROTEIN FOR BRAIN TUMORS

恶性脑瘤是最危及生命的癌症之一，而胶质母细胞瘤是最具侵袭性的脑癌。它约占所有原发脑肿瘤的35%。在美国，每年大约有17,500例新的脑瘤病例。如今，包括手术、放疗和化疗在内的标准疗法对患者存活率的影响相对较小。白介素4与假单胞菌外毒素(IL-4PE)融合产生的靶向性毒性蛋白可能为复发的胶质母细胞瘤提供一种新的治疗途径。PE是一种非常有效的毒素，当内化时，这种毒素会导致细胞死亡。恶性脑肿瘤表达丰富的IL-4受体，与IL-4具有很高的亲和力。因此，IL-4与PE的融合将提供一种通过内吞作用将外毒素传递到肿瘤细胞的有效途径。Neurocrine已经从FDA获得了IL-4PE的许可，并打算将这种融合蛋白开发成商业产品。要做到这一点，我们首先要构建一个能够高产表达该蛋白的表达载体。其次，我们将设计方法学对表达的蛋白质进行纯化和化学性质鉴定。第三，我们将建立体外细胞培养实验，以检测纯化的IL-4PE的效力。最后，我们将制备用于药代动力学研究的放射性标记的IL-4PE，并建立一种双点定向的ELISA法来检测动物和人体组织中的完整蛋白。这些信息将有助于我们将IL-4PE推进到第二阶段测试。建议的商业应用：目前治疗脑肿瘤的方法包括手术、放射治疗和化疗。然而，这些患者的预后仍然很差。IL-4与假单胞菌外毒素(IL-4PE)融合产生的靶向毒素可能为脑肿瘤患者提供一种有效的治疗方法。本申请中提出的研究将进一步推动这种新型疗法的发展。

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.