Glycoengineering eCD4-Ig

糖工程 eCD4-Ig

基本信息

批准号：
10326424
负责人：
MICHAEL DAVID ALPERT
金额：
$ 30万
依托单位：
EMMUNE, INC
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-14 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10326424
关键词：
Antibodies Area Asialoglycoprotein Receptor Binding Blood Circulation Carbohydrates Cell Line Chinese Hamster Ovary Cell Cyclic GMP DNA cassette Data Dose Drug Kinetics Engineering Enzymes FCGR3B gene Fucose Galactose Glucosamine 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 Peptide antibodies 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-dependent cell cytotoxicity base carbohydrate structure dimer glycosylation head-to-head comparison improved inhibiting antibody knock-down mouse model neonatal Fc receptor neutralizing antibody overexpression peptidomimetics prevent receptor binding sialylation small hairpin RNA stable cell line sugar therapeutic protein viral resistance

项目摘要

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.

抽象的我们正在开发 eCD4-Ig，这是一种由 CD4 构建的抗体样分子，CD4 是一种辅助受体模拟物肽和抗体 Fc，作为 HIV 的长效注射疗法。我们迄今为止的进展包括将 eCD4-Ig 的血浆半衰期延长至现在可与或超过的程度领先的广泛中和抗体 (bNAb) 正在开发为长效注射剂。在一个人 FcRn 转基因小鼠模型中药代动力学 (PK) 的头对头比较， eCD4-Ig 的 PK 明显优于在人体中半衰期为 71 天的 VRC01 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 条件下生产 eCD4-Ig。然而，深入分析其组成该细胞系产生的蛋白质 Fc 的 N297 处的 N 连接聚糖 (NLG) 突出显示了全领域共有的问题：绝大多数NLG都是不利于PK的形式。这问题在对抗体有活性的无岩藻糖基化形式中尤其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，我们建议对我们的细胞系进行糖工程用于生产 eCD4-Ig。为此，我们将开发一个糖工程基因盒，将其引入进入细胞系，并衍生出用于在 cGMP 下生产 eCD4-Ig 的糖工程克隆状况。尽管我们的主要目标是制造几乎完全由对于 ADCC 具有活性的单一长寿命糖型，可以使用相同的糖工程基因盒用于制造其他抗体疗法，包括 bNAb，以类似地扩展效应器功能和血浆半衰期。这项工作将降低治疗浓度并增加治疗间隔可以服用用于治疗和预防 HIV 感染的长效蛋白质疗法。