Discovery of Critical Host Genes Enabling Resistance to HIV-1 Infection as Therap

描述(申请人提供)：人类免疫缺陷病毒(HIV-1)是对公共健康的全球威胁，对药物开发构成前所未有的挑战。直接针对病毒的HIV-1药物由于迅速出现抗药性而经常失败。功能遗传学(FGI)使用了一种专利技术--随机纯合子基因扰动(RHGP)，并发现了新的宿主基因靶点，可以阻断HIV-1的感染，而不会对细胞生存产生明显的有害影响。与病毒靶点不同，依赖细胞靶点的治疗预计将保持对包括抗药性病毒在内的所有艾滋病毒变种的广谱疗效。此外，通过消除对病毒的选择压力，宿主靶向绕过了耐药性的发展。第一阶段研究中确定的靶点使用siRNA方法进行了验证，也表明它们通常是CXCR4和CCR5嗜性HIV1病毒感染所必需的。这些结果证明了FGI专有的rHGP技术在识别人类基因组中阻止病毒感染的宿主靶点(S)方面的能力，同时是无毒的。在第二阶段的提案中，我们将利用单抗疗法开发针对艾滋病毒的新型靶向干预。我们将重点关注一个特定的宿主靶点，“Robo1”，它对HIV-1的生命周期是必不可少的。为此，我们发现HIV-1感染会激活原代CD4+T淋巴细胞中Robo1的表达。因此，Robo1独特地出现在活的、产生病毒的细胞的表面。在人的PBMC检测中，Robo1抗体显示出抗HIV-1的活性。因此，通过抗体依赖的细胞毒性(ADCC)和/或补体依赖的细胞毒性(CDC)等正常宿主防御机制，Robo1可以被单抗靶向阻断HIV的生命周期和/或消除感染细胞。我们将进行基于噬菌体的人源单链抗体文库的筛选，以确定选择性识别HIV-1感染细胞但不识别未感染细胞的抗体候选。这些领先的ScFv候选抗体将被构建成全长的人IgG1抗体，并使用分子进化方法，候选领先抗体的结合亲和力将提高几个数量级。然后将评估它们的广谱抗病毒能力，包括直接抑制病毒复制和/或在体外介导ADCC和CDC效应，以及在人源化小鼠中的疗效。抗体铅(S)将通过基于细胞、动物和组织的交叉反应分析进行进一步审查。基于抗病毒活性和迭代改进，我们将提名一种主要的Robo1抗体候选用于IND使能研究。我们相信，选择性靶向HIV感染细胞的概念具有令人兴奋的前景，开发针对广泛HIV分离株的治疗性单抗是可行的。 与公共卫生相关：目前大多数艾滋病药物针对的是艾滋病毒，因此能够通过病毒突变而产生抗药性。第一阶段项目寻求确定人类宿主目标(而不是艾滋病毒目标)，以防止艾滋病毒在其生命周期中利用宿主的细胞机制。第二阶段的总体目标是利用第一阶段确定的目标开发面向宿主的治疗性抗体。这些抗体预计对艾滋病毒的所有变种都有效，包括那些对目前的抗病毒疗法有抵抗力的变种。

DESCRIPTION (provided by applicant): Human Immunodeficiency Virus (HIV-1) is a global threat to public health and poses an unprecedented challenge to drug development. HIV-1 drugs that directly target virus often fail due to rapid emergence of drug resistance. Functional Genetics (FGI) used a proprietary technology, Random Homozygous Gene Perturbation (RHGP) and discovered novel host gene targets that block HIV-1 infection without observable deleterious effect on cell survival. Unlike viral-targets, treatments relying on cellular targets are expected to retain broad spectrum efficacy against all HIV variants including drug-resistant viruses. In addition, by removing the selective pressure on the virus, host targeting circumvents drug resistance development. The identified targets in Phase I studies were validated using a siRNA approach and also shown to be commonly necessary for infection by both CXCR4 and CCR5 tropic HIV1 viruses. These results demonstrate the power of FGI proprietary RHGP technology to identify host targets(s) in human genome that block viral infection and at the same time are non-toxic. In the Phase II proposal, we will develop novel targeted intervention against HIV using monoclonal antibody therapeutics. We will focus on one particular host target, "Robo1", which is essential for the life cycle of HIV-1. To this end, we have discovered that Robo1 expression is activated in primary CD4+ T lymphocytes by HIV-1 infection. As a result, Robo1 uniquely appears on the surface of live, viral producing cells. Robo1 antibodies exhibited anti-HIV-1 activity in human PBMC assays. Robo1 thus can be targeted by monoclonal antibodies to block HIV life cycle and/or to eliminate infected cells via normal host defense mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). We will conduct phage-based screening of human scFv libraries to identify antibody candidates that selectively recognize HIV-1 infected but not uninfected cells. These leading scFv candidates will be built out into full- length human IgG1 antibodies and using molecular evolution approach, the binding affinities of lead antibody candidates will be enhanced by several orders of magnitude. Their broad spectrum anti-viral abilities will then be evaluated including direct inhibition of viral replication and/or mediation of ADCC and CDC effects in vitro and efficacy in humanized mice. Antibody lead(s) will be further scrutinized through cell-based, animal-based and tissue cross-reactivity assays. Based on antiviral activities and iterative improvements, we will nominate a lead Robo1 antibody candidate for IND-enabling studies. We believe the concept of selective targeting of HIV- infected cells has exciting prospects and that it is feasible to develop a therapeutic monoclonal antibody against a broad spectrum of HIV isolates. PUBLIC HEALTH RELEVANCE: Most current AIDS drugs target the HIV virus and therefore enable the development of drug resistance through viral mutation. The Phase I project seeks to identify human host targets (as opposed to HIV virus targets) that will prevent the HIV virus from using the host's cellular mechanism for its life cycle. The overall goal of Phase II is to develop Host Oriented Therapeutic antibodies using targets identified from Phase I. These antibodies are expected to be active against all variants of HIV, including those with resistance to current antiviral therapies.