Glycoengineering eCD4-Ig

糖工程 eCD4-Ig

• 批准号: 10589151
• 负责人: MICHAEL DAVID ALPERT
• 金额: $ 100万
• 依托单位: EMMUNE, INC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-09 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589151
• 关键词: Antibodies; Area; Asialoglycoprotein Receptor; Binding; Carbohydrates; Cell Line; Chinese Hamster Ovary Cell; Circulation; Cyclic GMP; DNA cassette; Data; Dose; Drug Kinetics; Engineering; Enzymes; FCGR3B gene; Fucose; Galactose; Glucosamine; Glycoengineering; Goals; HIV; HIV Infections; HIV Receptors; Half-Life; Human; Infection; Injectable; Link; Longevity; Macaca mulatta; Mannose; Mediating; Muscle; National Institute of Allergy and Infectious Disease; Pharmaceutical Preparations; Plasma; Polysaccharides; Primates; Property; Proteins; Recombinant Proteins; SIV; Sialic Acids; Testing; Therapeutic; Therapeutic antibodies; Time; Transgenic Mice; Treatment Efficacy; Viral Load result; Work; adeno-associated viral vector; antibody mimetics; antibody-dependent cell cytotoxicity; carbohydrate structure; dimer; glycosylation; head-to-head comparison; improved; inhibiting antibody; knock-down; manufacture; mouse model; neonatal Fc receptor; neutralizing antibody; overexpression; peptidomimetics; prevent; receptor; receptor binding; sialylation; small hairpin RNA; stable cell line; sugar; therapeutic protein; viral resistance

ABSTRACT We are developing eCD4-Ig, an antibody-like molecule constructed from CD4, a coreceptor-mimetic peptide, and an antibody Fc, as a long-acting injectable therapeutic for HIV. Our progress to-date includes having extended the plasma half-life of eCD4-Ig to the point that it now rivals or surpasses that of the leading broadly neutralizing antibodies (bNAbs) being developed as long-acting injectables. In a head-to-head comparison of pharmacokinetics (PK) in the human FcRn-transgenic mouse model, eCD4-Ig had significantly better PK than a VRC01 protein that had a half-life of 71 days in humans (Gaudinski et al., PLoS Med, 2018). With support from NIAID, we have developed a CHO cell line for manufacturing eCD4-Ig under cGMP conditions. However, in-depth analysis of the composition of the N-linked glycans (NLGs) at N297 of the Fc of the protein made by the cell line has highlighted a problem shared by the entire field: The large majority of the NLGs are forms detrimental to PK. This problem is particularly pronounced among the afucosylated forms that are active for antibody- dependent cell-mediated cytotoxicity (ADCC). Indeed, only a few percent of the NLGs are afucosylated forms that allow both ADCC and a long half-life. Therefore, we propose glycoengineering our cell line for manufacturing eCD4-Ig. To do so, we will develop a glycoengineering gene cassette, introduce it into the cell line, and derive a glycoengineered clone for manufacturing eCD4-Ig under cGMP conditions. Although our primary goal is to manufacture eCD4-Ig protein consisting almost entirely of a single long-lived glycoform that is active for ADCC, the same glycoengineering gene cassette can be used to manufacture other antibody therapeutics, including bNAbs, to similarly extend effector function and plasma half-life. This work will reduce the therapeutic concentration and increase the interval at which long-acting protein therapeutics for treating and preventing HIV infection can be dosed.

摘要 我们正在开发eCD 4-IG，这是一种由辅助受体模拟物CD 4构建的抗体样分子 肽和抗体Fc，作为HIV的长效注射治疗剂。我们迄今的进展 包括将eCD 4-IG的血浆半衰期延长到现在可以媲美或超过 领先的广泛中和抗体（bNAb）正在开发为长效注射剂。中 人FcRn转基因小鼠模型中药代动力学（PK）的头对头比较， eCD 4-IG的PK显著优于在人体中半衰期为71天的VRC 01蛋白 （Gaudinski等人，PLoS Med，2018）。在NIAID的支持下，我们开发了CHO细胞系， 在cGMP条件下制备eCD 4-IG。但是，深入分析了 由细胞系制备的蛋白质的Fc的N297处的N-连接聚糖（NLG）突出了由细胞系制备的蛋白质的Fc的N297处的N-连接聚糖（NLG）。 整个领域共有的问题：绝大多数NLG都是对PK有害的形式。这 问题在对抗体有活性的无岩藻糖基化形式中特别明显， 依赖性细胞介导的细胞毒性（ADCC）。事实上，只有百分之几的NLG是无岩藻糖基化的， 允许ADCC和长半衰期的形式。因此，我们提出糖工程我们的细胞系 用于制造eCD 4-IG。为此，我们将开发一种糖工程基因盒， 转化到细胞系中，并衍生用于在cGMP下生产eCD 4-IG的糖工程化克隆 条件虽然我们的主要目标是制造几乎完全由一种蛋白质组成的eCD 4-IG蛋白， 对于对ADCC有活性的单一长寿命糖型，相同的糖工程化基因盒可以是 用于生产其他抗体治疗剂，包括bNAb，以类似地延长效应子功能 和血浆半衰期。这项工作将降低治疗浓度并增加治疗间隔 可以给药用于治疗和预防HIV感染的长效蛋白质治疗剂。