Targeting the HIV reservoir using optimized anti-HIV antibodies

摘要 HIV-1的快速变异率导致了数千种病毒株，从而挫败了目前的疫苗 努力。虽然HIV-1感染可以通过抗逆转录病毒疗法(ART)控制，但病毒在 停止抗逆转录病毒治疗数周，因为潜伏的病毒库阻止了艾滋病毒-1的完全消除。大体上 从一个子集分离的针对HIV-1包膜尖峰(Env)的中和抗体(BNAbs) 感染艾滋病毒-1的捐赠者对艾滋病毒-1感染具有保护作用，并可以在感染后降低病毒载量，以及 有人提出，bNAbs可能在消除病毒库方面发挥作用。然而，HIV-1可以 即使是最强的bNAbs也可以通过突变来躲避。在这里，我们试图改造bNAbs来抵抗病毒 突变，这样它们就可以用来消灭病毒库。我们的战略依赖于利用贪婪效应 在个人和群体水平上预防病毒对bNAbs的耐药性。我们假设HIV-1病毒 阻止免疫球蛋白使用两种抗原结合纤维以二价键结合。这是由小规模的 HIV-1环境尖峰的数量和低密度，这阻止了大多数免疫球蛋白的尖峰间交联(双价 穗之间的结合)，以及阻碍穗内交联的环境三聚体的结构 (穗内的二价结合)。我们认为，主要的单价结合扩大了范围 HIV-1突变允许抗体逃避，而试剂能够通过尖峰内二价结合 多个HIV-1毒株之间的交联会更有效。这一假设得到了我们的 演示了我们的第一代Intra-2将几何平均效力提高了100倍 尖峰交联剂(通过刚性DNA连接物连接的同-和异-双纤维)。这些结果支持 假设HIV的低尖峰密度导致HIV-1 bNAbs对尖峰突变和 建议理想的抗艾滋病毒治疗方法将利用亲和力来实现体内 因为这种治疗方法会降低灭菌所需的浓度 对环境病毒的突变具有免疫力和抵抗力。在这里，我们建议设计、生产和评估第二个 产生尖峰内交联剂有两个改进：(I)它们将包含一个免疫球蛋白Fc来调节 效应器功能和增加血清半衰期，以及(Ii)DNA将被结构蛋白取代 链接器。我们还将评估旨在增强Fc介导的效应器的Fc替代的效果 通过更紧密地结合激活FcγR受体而发挥作用，并通过增强 与FcRN的结合，包括一种新的计算设计策略，以在条件下改善与FcRN的结合 促进了免疫球蛋白半衰期的增加。这些更有效的bNAbs可以用于治疗 从而减少了成本和/或生产时间，增加了接受治疗的病人数量， 并降低免疫原性或与使用bNAb相关的其他副作用的可能性。

Summary The rapid mutation rate of HIV-1 results in many thousands of viral strains, thus thwarting current vaccine efforts. Although HIV-1 infection can be controlled by anti-retroviral therapy (ART), the virus rebounds within weeks of ART cessation because complete elimination of HIV-1 is prevented by latent viral reservoirs. Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope spike (Env) that have been isolated from a subset of HIV-1–infected donors are protective against HIV-1 infection and can lower the viral load after infection, and it has been suggested that bNAbs could play a role in eliminating the viral reservoir. However, HIV-1 can evade even the most potent bNAbs by mutation. Here we seek to engineer bNAbs to be resistant to viral mutation so they could be used to eliminate viral reservoirs. Our strategy relies upon harnessing avidity effects to prevent viral resistance to bNAbs at both an individual and population level. We hypothesize that HIV-1 hinders IgGs from using both antigen-binding Fabs to bind bivalently. This is accomplished by the small number and low density of HIV-1 Env spikes, which prevent most IgGs from inter-spike crosslinking (bivalent binding between spikes), and the architecture of the Env trimer, which impedes intra-spike crosslinking (bivalent binding within a spike). We suggested that predominantly monovalent binding expands the range of HIV-1 mutations permitting Ab evasion, whereas reagents capable of bivalent binding through intra-spike crosslinking would be more potent across multiple strains of HIV-1. This hypothesis was supported by our demonstration of up to 100-fold increases in geometric mean potency achieved with our first generation intra- spike crosslinking reagents (homo- and hetero-diFabs joined by rigid DNA linkers). These results support the hypothesis that HIV's low spike density contributes to vulnerability of HIV-1 bNAbs to spike mutations and suggests that the ideal anti-HIV therapeutic for eliminating HIV reservoirs would utilize avidity to achieve intra- spike crosslinking because this sort of therapeutic would reduce the concentration required for sterilizing immunity and be resistant to Env mutations. Here we propose to design, produce, and evaluate second generation intra-spike crosslinking reagents with two improvements: (i) they will contain an IgG Fc to mediate effector functions and increase the serum half-life, and (ii) the DNA will be replaced by structured protein linkers. We will also evaluate the effects of Fc substitutions designed to enhance Fc-mediated effector functions through tighter binding to activating FcγR receptors and improve serum half-life through enhanced binding to FcRn, including a novel computational design strategy to improve binding to FcRn under conditions that promote increased IgG half-life. These more potent bNAbs could be used therapeutically at lower concentrations and thus reduce cost and/or production time, increase the number of patients being treated, and lower the potential for immunogenicity or other side-effects related to bNAb administration.