Data_Sheet_1_Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors.docx

The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens. Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH.

人类补体系统是免疫系统的重要组成部分，负责裂解并清除入侵的微生物以及凋亡体细胞。补体调节因子主要为H因子（factor H, FH）与H因子相关蛋白（FH-related proteins, FHRs），当其出现缺失、突变或自身抗体时，会引发补体系统异常激活，进而导致严重的肾脏与眼部疾病。然而目前市场上尚无重组FH治疗药物获批上市。首个成功实现FH重组表达的生物反应器系统为小立碗藓（Physcomitrella patens）苔藓生物反应器。近期，研究人员设计了人工合成调节因子MFHR1，将FH与H因子相关蛋白1（FH-related protein 1, FHR1）的活性相结合，构建出多靶点补体抑制剂。此前通过瞬时转染昆虫细胞的概念验证研究，已证实MFHR1的应用潜力。本研究首次实现在苔藓生物反应器中稳定生产经糖基化工程改造的重组MFHR1。该苔藓表达系统的核心优势包括：基于同源重组的精准基因组工程改造、符合药品生产质量管理规范（Good Manufacturing Practice, GMP）的光生物反应器规模化生产、极高的批次间重复性以及产物稳定性。目前已有多款潜在生物制药候选物通过该系统进行生产，在部分案例中此类产物甚至属于“生物改良型药物（biobetters）”：与哺乳动物表达系统生产的重组蛋白相比，苔藓表达系统制备的重组蛋白品质更优，例如已成功完成I期临床试验的苔藓源aGal。本研究通过基于质谱的分析手段，证实苔藓表达系统制备的MFHR1可正确合成并发生糖基化修饰，其糖链主要为复合型N-聚糖。苔藓源MFHR1展现出与FH相当的辅因子活性与衰变加速活性；其通过作用于补体激活替代途径的多个环节，对整条替代途径展现出强抑制效果，且抑制活性优于生理调节因子FH。