Structure-based expansion of neutralization ability of KD-247, an anti-V3 mAb

描述（由申请人提供）：抗hiv治疗在抑制hiv感染患者的病毒载量方面非常成功。然而，需要新的治疗策略来避免对所有目前使用的药物具有耐药性的病毒的不可避免的选择。在32种被批准用于治疗HIV感染的药物中，只有两种可以阻断病毒进入，而且目前还没有被批准的靶向HIV表面糖蛋白gp120的药物。KD-247是一种很有前途的人源单克隆抗体（mAb），由合作者松下修三博士及其同事开发。它结合gp120的V3环，并非常有效地中和广泛的多种B支HIV-1分离株。此外，KD-247还能抑制对所有目前批准的药物具有耐药性的病毒株的复制。基于其有前景的特性，KD-247已进入治疗HIV-1感染的Ib期临床试验。然而，KD-247与其他抗v3抗体一样，缺乏跨枝中和能力。KD-247进化支特异性的结构基础尚不清楚。该提案将侧重于使用基于结构的设计来构建具有更广泛进化特异性的KD-247的第二代单链可变片段（scFvs）。在初步结果中，我们以迄今为止所有人源化抗体报道的最高分辨率（1.55 E）解决了KD-247的非配体抗原结合片段（Fab）的晶体结构。我们还获得了一个基于野生型KD-247的克隆scFv，用于突变体的构建，并进行了分子建模研究，这将初步指导scFv突变的选择。我们建议扩展这些研究，以追求以下具体目标：为了确定为什么KD-247能有效中和广谱的B支分离株2。设计、构建和评估有效结合B支系和非B支系分离株的scFv KD-247。实现本提案的目标将提供结构、生化和病毒学知识，指导第二代抗v3抗体的设计和工程，具有更高的效力和更广泛的中和能力。此外，我们预期的高分辨率结构也将提供分子蛋白-蛋白质接触的基本信息，这对抗体识别特定抗原很重要。基于结构的抗hiv单抗工程是一种创新方法，可以作为设计其他治疗方法的范例。

DESCRIPTION (provided by applicant): Anti-HIV treatments have been very successful in suppressing viral load in HIV-infected patients. However, new therapeutic strategies are needed to circumvent the inevitable selection of viruses that are resistant to all currently used drugs. Of the 32 drugs approved for the treatment of HIV infection, only two block viral entry, and there are no approved drugs that target the HIV surface glycoprotein gp120. KD-247 is a promising humanized monoclonal antibody (mAb) that has been developed by collaborator Dr. Shuzo Matsushita and colleagues. It binds the V3 loop of gp120 and neutralizes extremely potently a broad panel of diverse clade B HIV-1 isolates. Moreover, KD-247 suppresses replication of viral strains that are resistant to all currently approved drugs. Based on its promising properties, KD-247 has entered Phase Ib clinical trials for the treatment of HIV-1 infections. However, KD-247 like other anti-V3 antibodies lacks cross- clade neutralization ability. The structural basis for the clade specificity of KD-247 is unclear. This proposal will focus on the use of structure-based design to construct second generation single-chain variable fragments (scFvs) of KD-247 with broader clade specificity. In preliminary results, we have solved the crystal structure of the unliganded antigen-binding fragment (Fab) of KD-247 at the highest resolution (1.55 E) reported for any humanized antibody to date. We have also obtained a cloned scFv based on wild-type KD-247 to be used for the construction of mutants and performed molecular modeling studies that will initially guide the selection of scFv mutations. We propose to extend these studies to pursue the following specific aims: 1. To determine why KD-247 neutralizes efficiently a broad spectrum of clade B isolates 2. To design, construct, and evaluate scFv KD-247 that effectively bind clade B AND non-clade B isolates Achieving the aims of this proposal will provide structural, biochemical, and virological knowledge that will direct the design and engineering of second generation anti-V3 antibodies with improved potency and broader neutralization ability. In addition, our expected high-resolution structures will also provide essential information about molecular protein-protein contacts important for antibody recognition of specific antigens. Structure-based anti-HIV mAb engineering is an innovative approach that can serve as a paradigm for the design of additional therapeutics. PUBLIC HEALTH RELEVANCE: This project will determine crystal structures of KD-247, a monoclonal antibody currently in clinical trials, that will help us understand why it works well with many, but not all types of HIV. This knowledge will help us design second generation antibodies with broader neutralization ability.