Engineering of broadly reactive seroantibodies

背景/原理：目前，还没有有效的人类免疫缺陷病毒预防性疫苗 (艾滋病毒)-1，也没有治愈这种慢性感染的方法。至少每250名退伍军人中就有一人感染艾滋病毒。HIV-1 B分支菌株是美国的主要菌株，也是退伍军人的压倒性感染源 (97%为支系B)。唯一可用的治疗方法是终身治疗和联合抗逆转录病毒治疗。作为一部分 在我们目前的荣誉奖中，我们成功地发现了一种新的广泛中和抗体家族 (BNAbs)是迄今为止描述的最有效和最广泛的(N49 P系列)，包括N49P7(100%广度 在117病毒组中，IC50中值为0.10 ug/ml)和N4P9.3(117病毒组中97%的广度，IC50中值为 0.0495微克/毫升)。此外，我们从同时循环的分支B中鉴定了HIV-1包膜序列 来自同一捐赠者的病毒，从而描绘出它们的抗药性突变。其他初步数据包括 展示抗体工程的电子数据，以及展示所用小鼠模型的活体数据。 目的：我们的假设是，我们可以利用抗体和病毒被膜序列来设计 N49 P系列bNAbs走向更大的广度和效力，从而涵盖具有潜在 逃避自然产生的N49的bNAbs和类似的特异性。这项建议的具体目标是1) 设计N49P系列bNAbs以优化临床应用；和2)在两个阶段测试工程bNAbs 人源化小鼠模型以建立体内对抗HIV-1的效力。 方法：我们的计划是首先从N49P系列bNAbs中开发一组工程化的60 mAbs。 工程师将从有关野生型N49P7和N49P9.3的结晶学信息中了解Fab- Gp120相互作用；在标准中发现的N49P7和N49P9.3抗性环境变异体的电子模拟中 筛选板；在对N49P7和N49P9.3抗性环境变异体的电子模拟中，在 含有bNAb血统的同时代血浆。目标将是确定关键残留物 参与中和(重点是分支B菌株)，以及那些不利于稳定的菌株。 有了这些知识，我们将创造60个突变体，目的是提高效力/广度，同时保持或 提高稳定性，缺乏自动/多反应性，以及ADCC。5个最终bNAb(LS格式)的列表将在 人源化小鼠模型的半衰期和体内疗效(无细胞的自体和异体挑战 以及HU-PBL小鼠模型中的细胞相关挑战)。 发现：预期的结果将是从N49 P系列家族中分离出新的工程bNAbs，这将 表现出更好的效力和广度，以及足够的稳定性和PK。此外，这些bNAb将是 在人源化小鼠模型中进行体内疗效测试，进一步将这些产品推向临床使用。 现状：我们已经分离出N49P系列的bNAbs。我们组建的团队在 单抗生产、HIV-1病毒测序、生物化学和抗体工程以及小鼠 HIV-1感染模型。 影响：如果我们的假设被证明是正确的，这些bNAbs将代表着临床上的突破和领先 候选抗体正被考虑用于HIV-1的预防和治疗，并可用于各种 尤其是在退伍军人医院，因为艾滋病毒仍然是我们退伍军人的一个问题。

Background/Rationale: Currently, there is no effective preventative vaccine for human immunodeficiency virus (HIV)-1, nor is there a cure for this chronic infection. At least one in 250 Veterans is infected with HIV. HIV-1 Clade B strains are the predominant strain in the US, and the overwhelming source of infection for Veterans (97% Clade B). The only treatment available is life-long therapy with combined anti-retroviral therapy. As part of our current Merit Award, we were successful in discovering a new family of broadly neutralizing antibodies (bNAbs) that are the most potent and broad described to date (N49 P series), including N49P7 (100% breadth in 117 virus panel, median IC50 0.10 ug/ml) and N4P9.3 (97% breadth in the 117 virus panel, median IC50 0.0495 ug/ml). Further, we identified HIV-1 envelope sequences from contemporaneous, circulating Clade B viruses from the same donor, thus delineating their resistance mutations. Other preliminary data include in silico data demonstrating antibody engineering, and in vivo data demonstrating the mouse models used. Objectives: Our hypothesis is that we can exploit both antibody and viral envelope sequences to engineer the N49 P series bNAbs towards even greater breadth and potency, thus covering variants with the potential to escape naturally occurring bNAbs of N49 and similar specificities. The specific aims of this proposal are 1) Engineer the N49 P series bNAbs to optimize clinical utility; and 2) Test the engineered bNAbs in two humanized mice models to establish in vivo efficacy against HIV-1. Methods: Our plan is to first develop a panel of engineered 60 mAbs from the N49 P series bNAbs. Engineering will be informed by crystallographic information regarding wild type N49P7 and N49P9.3 Fab- gp120 interactions; in silico modeling of N49P7 and N49P9.3 resistant Env variants found in standard screening panels; in silico modeling of N49P7 and N49P9.3 resistant Env variants found in the contemporaneous plasma that harbored the bNAb lineages. The aim will be to determine the key residues that are involved in neutralization (with a focus on Clade B strains), as well as those that are detrimental to stability. With this knowledge, we will create 60 mutants with the aim of improving potency/breadth, while maintaining or improving stability, lack of auto/polyreactivity, and ADCC. A list of 5 final bNAbs (in LS format) will be tested in humanized mice models for half-life, and in vivo efficacy (autologous and heterologous challenges in cell-free and cell-associated challenges in the Hu-PBL mouse model). Findings: Anticipated results will be isolation of new engineered bNAbs from the N49 P series family that will demonstrate improved potency and breadth, and adequate stability and PK. Furthermore, these bNAbs will be tested in a humanized mouse models for in vivo efficacy, further moving these products toward clinical use. Status: We have isolated the N49 P series of bNAbs. The team we have assembled has expertise in monoclonal antibody production, HIV-1 virus sequencing, biochemistry and antibody engineering, and mouse models of HIV-1 infection. Impact: If our hypothesis proves correct, these bNAbs will represent a clinical breakthrough and the lead candidate for antibodies being considered for HIV-1 prophylaxis and treatment, and could be used in a variety of clinical formats, especially at the VA, as HIV continues to be a problem for our Veterans.