ABSTRACT

Broadly neutralizing antibodies are showing promise in the treatment and prevention of HIV-1, with several now being evaluated clinically. Some lead clinical candidates, including antibodies CAP256-VRC26.25, N6, PGT121, and VRC07-523, have one or more N-linked glycosylation sequons in their variable domains (Fvs) from somatic hypermutation, and these glycans increase chemical heterogeneity, complicating the manufacture of these antibodies as products. Here we propose a general method to remove Fv glycans and use this method to develop engineered versions of these four antibodies with Fv glycans removed. When germline residues were introduced to remove each glycan, antibody properties between wild type and mutant were not significantly altered for CAP256-VRC26.25 and PGT121; however, germline mutants for N6 and VRC07-523 showed increased polyreactivity, which is known to correlate with unfavorable in vivo pharmacokinetics. To reduce polyreactivity induced by removal of Fv glycan, we mutated aromatic residues and arginines structurally proximal to the removed glycan and identified Fv glycan-removed variants with low polyreactivity for N6 and VRC07-523. Two such variants, N6-N72_LCQ-R18_LCD and VRC07-523-N72_LCQ-R24_LCD, showed thermostability, neutralization potency and breadth, and half-life in humanized FcRn mice that were similar to their wild-type Fv-glycosylated counterparts. The removal of Fv glycan and reduction of chemical heterogeneity were confirmed by liquid chromatography-mass spectrometry. With reduced heterogeneity, the Fv-glycan-removed variants developed here may have utility as products for treating or preventing infection by HIV-1.

摘要

广泛中和抗体在 HIV-1 的治疗和预防方面显示出前景，目前有几种抗体正在进行临床评估。一些主要的临床候选药物，包括抗体 CAP256-VRC26.25、N6、PGT121 和 VRC07-523，具有一个或多个N来自体细胞超突变的可变域 (Fvs) 中的 -连接的糖基化序列，这些聚糖增加了化学异质性，使这些抗体作为产品的制造复杂化。在这里，我们提出了一种去除 Fv 聚糖的通用方法，并使用这种方法开发这四种抗体的工程版本，其中去除了 Fv 聚糖。当引入种系残基以去除每个聚糖时，野生型和突变体之间的抗体特性对于 CAP256-VRC26.25 和 PGT121 没有显着改变；然而，N6 和 VRC07-523 的种系突变体表现出增加的多反应性，这与体内不利相关药代动力学。为了减少去除 Fv 聚糖引起的多反应性，我们突变了结构上与去除的聚糖接近的芳香族残基和精氨酸，并鉴定了 N6 和 VRC07-523 具有低多反应性的 Fv 聚糖去除变体。两个这样的变体，N6-N72 _LC Q-R18 _LC D 和 VRC07-523-N72 _LC Q-R24 _LCD，在人源化 FcRn 小鼠中显示出热稳定性、中和效力和广度以及半衰期，与它们的野生型 Fv 糖基化对应物相似。液相色谱-质谱法证实了 Fv 聚糖的去除和化学异质性的减少。由于异质性降低，这里开发的去除 Fv-聚糖的变体可用作治疗或预防 HIV-1 感染的产品。