Manufacturing of Trojan Horse-TNFR Decoy Receptor Fusion Protein

描述(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发挥促炎作用，如阿尔茨海默病或帕金森病。生物肿瘤坏死因子抑制物(TNFi)，如肿瘤坏死因子诱骗受体，不能用于治疗脑部疾病，因为TNFI是不能通过血脑屏障(BBB)的大分子。目前的工作是对穿透血脑屏障的生物TNFi的药物开发，它是人类II型肿瘤坏死因子受体(TNFR)的重新设计形式，其中TNFR作为一种免疫球蛋白融合蛋白产生。Ig G部分是一种针对人胰岛素受体(HIR)的基因工程单抗(MAb)。HIRMAb-TNFR融合蛋白命名为AGT-110。HIRMAb-TNFR融合蛋白的HIRMAb部分作为一个分子特洛伊木马，通过内源性BBB胰岛素受体上的受体介导的运输，将融合的诱骗受体运送到BBB。在二期SBIR项目中，将开发适合于临床试验生产的HIRMAb-TNFR融合蛋白的大规模制造方法，以及大量研究药物。将稳定表达的宿主细胞培养在50L生物反应器中，用1L柱层析或蛋白A亲和层析、阳离子交换层析和阴离子交换层析纯化融合蛋白。药品的特性、纯度、效力、安全性和杂质将通过&gt；15测试方法进行评估。在最初的剂量范围研究中，将首次在成年恒河猴身上测试药物动力学、免疫反应、安全药理学和毒理学。如果成功，这项研究将为与FDA举行IND前会议以及AGT-110药物开发进入GLP药理学和GMP生产阶段提供基础。这项工作的总体目标是开发脑穿透性生物肿瘤坏死因子，以便抑制肿瘤坏死因子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.