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（如阿尔茨海默病或帕金森病）中起促炎作用。生物TNF抑制剂（TNFI），如TNF诱饵受体，不能用于脑部疾病，因为TNFI是不能穿过血脑屏障（BBB）的大分子。本工作继续BBB穿透生物TNFI的药物开发，TNFI是人II型TNF受体（TNFR）的再工程形式，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部分充当分子特洛伊木马，通过内源性BBB胰岛素受体上的受体介导的转运将融合的诱饵受体运送穿过BBB。在该II期SBIR项目中，将开发适用于生产研究药物临床试验批次的大规模生产HIRMAb-TNFR融合蛋白的方法。将稳定转染的宿主细胞在50 L生物反应器中培养，并将融合蛋白用1升柱或蛋白A亲和层析、阳离子交换层析和阴离子交换层析纯化。将通过>15种检测方法评价制剂的鉴别、纯度、效价、安全性和杂质。在初始剂量范围探索研究中，将首次在成年恒河猴中检测药代动力学、免疫应答、安全药理学和毒理学。如果成功，这项研究将为FDA的Pre-IND会议提供基础，并将AGT-110药物开发进入GLP药理学和GMP生产阶段。这项工作的总体目标是开发脑穿透生物TNFI，以便抑制TNF α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.