Manufacturing of Trojan Horse-TNFR Decoy Receptor Fusion Protein

描述（由申请人提供）：肿瘤坏死因子(TNF)- α在衰老相关的神经退行性疾病中起促炎作用，如阿尔茨海默病或帕金森病。生物TNF抑制剂（TNFI），如TNF诱饵受体，不能开发用于脑部疾病，因为TNFI是大分子，不能穿过血脑屏障（BBB）。目前的工作继续开发一种穿透血脑屏障的生物TNFI，这是一种人类II型TNF受体（TNFR）的重组形式，其中TNFR作为IgG融合蛋白产生。IgG部分是针对人胰岛素受体（HIR）的基因工程单克隆抗体（MAb）。HIRMAb-TNFR融合蛋白被命名为AGT-110。HIRMAb- tnfr融合蛋白的HIRMAb部分作为分子特洛伊木马，通过内源性血脑屏障胰岛素受体受体介导的转运，将融合的诱饵受体运送过血脑屏障。在这个二期SBIR项目中，将开发大规模生产适合临床试验大量研究药物的HIRMAb-TNFR融合蛋白的方法。稳定转染后的宿主细胞在50L生物反应器中培养，用1 l柱或蛋白a亲和层析、阳离子交换层析、阴离子交换层析纯化融合蛋白。药品的鉴别、纯度、效力、安全性和杂质将采用bbb15检验方法进行评价。在初始剂量范围研究中，将首次在成年恒河猴身上测试药代动力学、免疫反应、安全药理学和毒理学。如果成功，该研究将为与FDA的Pre-IND会议提供基础，并为AGT-110药物开发进入GLP药理学和GMP制造阶段提供基础。本工作的总体目标是开发脑穿透性生物TNFI，从而抑制TNFalpha在神经疾病中的促炎作用。

DESCRIPTION (provided by applicant): Tumor necrosis factor (TNF)-alpha plays a pro-inflammatory role in aging-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. The biologic TNF inhibitors (TNFI), such as the TNF decoy receptor, cannot be developed for brain diseases, because the TNFIs are large molecules that do not cross the blood-brain barrier (BBB). The present work continues the drug development of a BBB-penetrating biologic TNFI, which is a re-engineered form of the human type II TNF receptor (TNFR), wherein the TNFR is produced as an IgG fusion protein. The IgG part is a genetically engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb-TNFR fusion protein is named AGT-110. The HIRMAb part of the HIRMAb-TNFR fusion protein acts as a molecular Trojan horse to ferry the fused decoy receptor across the BBB via receptor-mediated transport on the endogenous BBB insulin receptor. In this phase II SBIR project, the methodology for manufacturing of the HIRMAb-TNFR fusion protein at a large scale suitable for production of clinical trial lots of study drug will be developed. The stably transfected host cell will be cultured in a 50L bioreactor, and the fusion protein will be purified with 1 liter columns or protein A affinity chromatography, cation exchange chromatography, and anion exchange chromatography. The identity, purity, potency, safety, and impurities of the drug product will be evaluated by >15 test methods. The pharmacokinetics, immune response, safety pharmacology, and toxicology will be tested for the first time in adult Rhesus monkeys in an initial dose-ranging study. If successful, this research will provide the basis for a Pre-IND Meeting with the FDA, and entry of AGT-110 drug development into the phases of GLP pharmacology and GMP manufacturing. The overall goal of this work is the development of brain penetrating biologic TNFI, so that the pro-inflammatory effects of TNFalpha in neural disease can be suppressed.