Targeting the HIV reservoir using optimized anti-HIV antibodies

总结 HIV-1的快速突变率导致成千上万的病毒株，从而阻碍了目前的疫苗 努力虽然HIV-1感染可以通过抗逆转录病毒疗法（ART）得到控制，但病毒会在体内反弹。 停止抗逆转录病毒治疗数周，因为潜伏的病毒库阻止了HIV-1的完全消除。广泛 已从一个子集中分离的抗HIV-1包膜刺突（Env）的中和抗体（bNAb） 的HIV-1感染供体对HIV-1感染具有保护作用，并可降低感染后的病毒载量， 已经提出bNAb可以在消除病毒储库中起作用。HIV-1可以 通过突变来逃避最强的bNAbs在这里，我们寻求工程化bNAb以抵抗病毒 这样它们就可以用来消除病毒储存库。我们的策略是利用贪婪效应 以防止在个体和群体水平上对bNAb的病毒抗性。我们假设HIV-1 阻碍IgG使用两种抗原结合Fab结合生物素。这是由小 HIV-1 Env刺突的数量和低密度，可防止大多数IgG刺突间交联（二价 刺突之间的结合），以及阻碍刺突内交联的Env三聚体的结构 （刺突内的二价结合）。我们认为，主要是单价结合扩大了范围 允许Ab逃避的HIV-1突变，而能够通过内加标结合的试剂 交联在HIV-1的多个菌株中将更有效。这一假设得到了我们的支持。 证明了我们的第一代细胞内- 加标交联试剂（通过刚性DNA接头连接的同源和异源diFab）。这些结果支持这一 假设HIV的低峰密度导致HIV-1 bNAb对峰突变的脆弱性， 表明消除艾滋病毒宿主的理想抗艾滋病毒治疗方法将利用亲合力来实现内- 因为这种治疗剂会降低消毒所需的浓度 免疫力和对Env突变的抗性。在这里，我们建议设计，生产和评估第二 具有两个改进的第二代加标物内交联试剂：（i）它们将含有IgG Fc以介导 效应子功能并增加血清半衰期，和（ii）DNA将被结构化蛋白质取代 连接器。我们还将评估设计用于增强Fc介导的效应子的Fc置换的作用。 通过更紧密地结合活化FcγR受体发挥作用，并通过增强Fc γ R受体的活性来改善血清半衰期。 与FcRn的结合，包括在一定条件下改善与FcRn结合的新型计算设计策略 促进IgG半衰期增加。这些更有效的bNAb可以在较低的温度下用于治疗。 因此降低了成本和/或生产时间，增加了治疗的患者数量， 并降低与bNAb给药相关的免疫原性或其它副作用的可能性。

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.