Glycoengineering eCD4-Ig

摘要 我们正在开发eCD4-Ig，一种由辅助受体模拟物CD4构建的类似抗体的分子 多肽和抗体Fc，作为HIV的长效注射疗法。我们到目前为止的进展 包括将eCD4-Ig的血浆半衰期延长到现在可以与之匹敌或超过的程度 领先的广谱中和抗体(BNAbs)正被开发为长效注射剂。在一个 人FcRN转基因小鼠模型药代动力学(PK)的面对面比较 ECD4-Ig的PK明显好于半衰期为71天的VRC01蛋白 (Gaudinski等人，PLoS Med，2018年)。在NIAID的支持下，我们已经开发出一种CHO细胞系，用于 在cGMP条件下制备eCD4-Ig。然而，深入分析 该细胞系制造的蛋白质的N297位的N-连接糖链(NLGS)突出了一种 整个领域的共同问题：绝大多数NLGS都是不利于PK的形式。这 这个问题在对抗体有活性的果糖形式中尤为明显- 依赖细胞介导的细胞毒作用(ADCC)。事实上，只有几个NLGS是果糖基化的。 既允许ADCC又允许长半衰期的形式。因此，我们建议对我们的细胞系进行糖工程。 用于制造eCD4-Ig。为此，我们将开发一种糖工程基因盒，介绍它 进入细胞系，并获得用于在cGMP作用下制造eCD4-Ig的糖工程克隆 条件。虽然我们的主要目标是制造eCD4-Ig蛋白，几乎完全由 对ADCC具有活性的单一长寿命糖型，相同的糖工程基因盒可以 用于制造其他抗体疗法，包括bNAbs，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，增加治疗间隔时间 治疗和预防艾滋病毒感染的长效蛋白质疗法可以用哪种。

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.