Manufacturing of Trojan Horse-TNFR Decoy Receptor Fusion Protein

描述（由申请人提供）：肿瘤坏死因子(TNF)-¿在脑部疾病中起促炎作用。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）。目前的工作继续进行人类II型肿瘤坏死因子受体（TNFR）的重新工程形式的药物开发，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。本研究的pre-SBIR可行性阶段描述了HIRMAb-TNFR融合蛋白的工程、表达、生化验证、体内血浆药代动力学和恒河猴血脑屏障转运。拟议的I期SBIR研究将制定生产HIRMAb-TNFR融合蛋白的生产计划。该生产将被设计用于生产纯度、效力、安全性和杂质方面符合FDA规范的治疗产品，以便在未来用于临床试验的融合蛋白的GMP生产中可以重复生产。在I期开发的小规模生产方法将在II期扩大到50L生物反应器阶段，用于生产可支持未来临床试验的HIRMAb-TNFR融合蛋白。

DESCRIPTION (provided by applicant): Tumor necrosis factor (TNF)-¿ plays a pro-inflammatory role in brain diseases. 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 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 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. The pre-SBIR feasibility stage of this research describes the engineering, expression, biochemical validation, and in vivo plasma pharmacokinetics and BBB transport in the Rhesus monkey of the HIRMAb-TNFR fusion protein. The proposed phase I SBIR research will develop a manufacturing scheme for production of the HIRMAb-TNFR fusion protein. This manufacturing will be designed to produce a therapeutic product that meets FDA specifications with regard to purity, potency, safety, and impurities, so that the manufacturing can be replicated in future GMP production of the fusion protein for clinical trials. The small scale manufacturing methodology developed in phase I will then be scaled up in phase II to the 50L bioreactor stage for manufacturing of the HIRMAb-TNFR fusion protein at production levels that can support future clinical trials. PUBLIC HEALTH RELEVANCE: Biologic tumor necrosis factor inhibitors (TNFI), such as the tumor necrosis factor (TNF) decoy receptor, cannot be developed for brain diseases, because these large molecule drugs do not cross the blood-brain barrier (BBB). The present research will re-engineer the human type II TNF receptor (TNFR) as an IgG-TNFR fusion protein, where the IgG part is a genetically engineered monoclonal antibody that crosses the BBB via transport on the endogenous insulin receptor. The IgG acts as a molecular Trojan horse to ferry across the BBB the TNFI.