Engineering of pan-neutralizing anti-HIV envelope antibodies

1背景/原理：一种越来越受关注的替代暴露前预防方式是基于 2前提是预防可以安全地实现被动免疫与广泛中和抗艾滋病毒 3种针对HIV包膜的抗体（bNAbs）。泛中和抗体可以提供一种可行的方法， 4.在全球范围内预防艾滋病毒感染。 5目的：我们的假设是，通过合理工程化接近泛中和的bNAb， 6结合位点（CD 4 bs）bNAbs N49 P7和N49P9.3，使用HIV的罕见抗性变体作为指导， 7中和，具有临床相关的药代动力学特征。这种泛中和抗体 8甚至将涵盖对其他抗CD 4 b bNAb（3BNC 117和N6）具有交叉抗性的变体。我们有 9初步数据表明N49 P7和N49P9.3接近泛中和，并表现出新的结构 10个特征（例如，N49 P7可进入高度保守的内部结构域），其可用于指导改进的 11 bNAb设计。这些独特的功能提供了最接近的起点，建立一个高效的泛- 12中和抗体。与此同时，我们的合作者已经开发了工程化bNAb的技术， 13的效力PGT 121约50倍，同时保留有利的品质（溶解性和非溶解性）。 14自体/多反应性）。本建议书的具体目标是：1）工程师N49 P7和N49P9.3， 2）来自Aim 1的bNAb变体的向下选择 3）在两个细胞中测试工程化的N49 bNAb; 17人源化小鼠模型使用耐药病毒，以确定其在预防HIV-1感染中的功效。 18方法：我们的计划是收集具有更大效力和广度的N49 P7和N49P9.3变体 19使用酵母表面展示，专注于选择具有增强的重组识别的变体。 20个产生了来自中和抗性病毒的gp 120和SOSIP。带输入的组合库 21从x射线晶体学，抗体谱系分析，中和逃逸残留物，和责任分析， 第22章也是设计重点将放在减少自动/多反应性能， 23针对那些结合膜蛋白的阴性选择，免疫原性预测算法，和 24个生殖系回复。经过3轮工程改造，我们将产生48株含有“LS”的人IgG 1单抗。 25半衰期延长，并评估bNAb的体外中和。此后，将检测8个bNAb， 26自身/多反应性、表达滴度、生物化学行为和体内半衰期。排名前两位的bNAb将是 在人源化小鼠模型中测试了27种抗无细胞和细胞相关HIV攻击的能力。 28影响：开发具有理想PK和溶解度特性的单一、完全泛中和抗体 缺乏可辨别的多反应性将是开发免疫治疗方法的一个重大进展。 30基于艾滋病毒的治疗和预防。

1 Background/Rationale: An alternative pre-exposure prophylaxis modality of growing interest is based on the 2 premise that prevention can be safely achieved by passive immunization with broadly neutralizing anti-HIV 3 antibodies (bNAbs) against the HIV envelope. A pan-neutralizing antibody could provide a feasible means to 4 prevent HIV infection worldwide. 5 Objectives: Our hypothesis is that by rational engineering of near pan-neutralizing bNAbs against the CD4 6 binding site (CD4bs) bNAbs N49P7 and N49P9.3, using rare resistance variants of HIV as guides, a pan- 7 neutralizing with clinically relevant pharmacokinetic profiles can be made. Such a pan-neutralizing antibody 8 would even cover variants that are cross-resistant to other anti-CD4bs bNAbs (3BNC117 and N6). We have 9 preliminary data demonstrating that N49P7 and N49P9.3 are near pan-neutralizing and exhibit novel structural 10 features (e.g., N49P7 has access to the highly conserved inner domain) that can be used to guide improved 11 bNAb design. These unique features provide the closest possible starting point for building a highly potent pan- 12 neutralizing antibody. In parallel, our collaborators have developed techniques to engineer bNAbs, increasing 13 the potency of PGT121 approximately 50 times, while retaining favorable qualities (solubility and non- 14 auto/polyreactivity). The specific aims of this proposal are 1) Engineer N49P7 and N49P9.3 to improve 15 neutralization of HIV-1 viruses resistant to CD4bs bNAbs; 2) Downselection of bNAb variants from Aim 1 16 based on in vivo pharmacokinetics and biophysical properties; 3) Test engineered N49 bNAbs in two 17 humanized mouse models using resistant viruses to determine their efficacy in preventing HIV-1 infection. 18 Methods: Our plan is to make a collection of N49P7 and N49P9.3 variants with greater potency and breadth 19 using yeast surface display, focusing on selecting variants with enhanced recognition of recombinantly 20 produced gp120s and SOSIPs derived from neutralization resistant viruses. Combinatorial libraries with input 21 from x-ray crystallography, antibody lineage analysis, neutralization escape residues, and liability analysis will 22 also inform design. Emphasis will be placed on reduction of auto/polyreactive properties by aggressively 23 negatively selecting against those binding membrane proteins, immunogenic prediction algorithms, and 24 germline reversion. After 3 rounds of engineering, we will produce 48 mAbs as human IgG1 containing the “LS” 25 half-life extension and evaluate the bNAbs for in vitro neutralization. Thereafter, 8 bNAbs will be tested for 26 auto/polyreactivity, expression titer, biochemical behavior, and in vivo half-life. The top two bNAbs will be 27 tested in humanized mouse models for ability to protection against cell-free and cell-associated HIV challenge. 28 Impact: The development of a single, fully pan-neutralizing antibody with desirable PK and solubility properties 29 absent discernable polyreactivity would be a major advance in efforts to develop approaches for immune- 30 based treatment and prevention of HIV.