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

描述（由申请人提供）：人类免疫缺陷病毒（HIV-1）是对公共卫生的全球威胁，对药物开发构成了前所未有的挑战。直接靶向病毒的HIV-1药物往往由于迅速出现耐药性而失败。功能遗传学（FGI）使用一种专有技术，随机纯合基因扰动（RHGP），发现了新的宿主基因靶点，可以阻断HIV-1感染，而不会对细胞存活产生明显的有害影响。与病毒靶点不同，依赖于细胞靶点的治疗预计将保留对所有HIV变体（包括耐药病毒）的广谱疗效。此外，通过消除对病毒的选择性压力，宿主靶向避免了耐药性的发展。在I期研究中鉴定的靶点使用siRNA方法进行了验证，并且也显示出对于嗜CXCR 4和CCR 5的HIV 1病毒的感染通常是必需的。这些结果证明了FGI专有RHGP技术鉴定人类基因组中阻断病毒感染同时无毒的宿主靶标的能力。 在第二阶段的建议中，我们将开发新的针对艾滋病毒的单克隆抗体疗法的干预措施。我们将重点关注一个特定的宿主靶点“Robo 1”，它对HIV-1的生命周期至关重要。为此，我们已经发现，Robo 1的表达被激活的主要CD 4 + T淋巴细胞的HIV-1感染。因此，Robo 1独特地出现在活的病毒产生细胞的表面。Robo 1抗体在人PBMC测定中表现出抗HIV-1活性。因此，Robo 1可以被单克隆抗体靶向，以阻断HIV生命周期和/或通过正常的宿主防御机制（如抗体依赖性细胞毒性（ADCC）和/或补体依赖性细胞毒性（CDC））消除感染的细胞。我们将进行基于噬菌体的筛选人类单链抗体库，以确定候选抗体，选择性地识别HIV-1感染，而不是未感染的细胞。这些先导scFv候选物将构建成全长人IgG 1抗体，并且使用分子进化方法，先导抗体候选物的结合亲和力将增强几个数量级。然后将评价它们的广谱抗病毒能力，包括直接抑制病毒复制和/或介导体外ADCC和CDC效应以及在人源化小鼠中的功效。将通过基于细胞、基于动物和组织的交叉反应性试验进一步检查抗体先导化合物。基于抗病毒活性和迭代改进，我们将提名一种领先的Robo 1抗体候选物用于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，并且开发针对广谱HIV分离株的治疗性单克隆抗体是可行的。 公共卫生相关性：目前大多数艾滋病药物针对HIV病毒，因此通过病毒突变使耐药性产生。第一阶段的项目旨在确定人类宿主的目标（而不是艾滋病毒的目标），这将防止艾滋病毒利用宿主的细胞机制，其生命周期。II期的总体目标是使用I期鉴定的靶标开发宿主导向的治疗性抗体。预计这些抗体对所有HIV变体都有活性，包括对当前抗病毒疗法具有抗性的变体。

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.