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规避对所有目前使用的药物都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，并且没有被批准的药物靶向HIV表面糖蛋白gp 120。 KD-247是一种很有前途的人源化单克隆抗体（mAb），由合作者Shuzo Matsushita博士及其同事开发。它与gp 120的V3环结合，并能非常有效地中和多种进化枝B HIV-1分离株。此外，KD-247抑制对所有目前批准的药物具有耐药性的病毒株的复制。基于其有前途的特性，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抗体一样缺乏交叉进化枝中和能力。 KD-247的进化枝特异性的结构基础尚不清楚。本研究旨在利用基于结构的设计方法构建KD-247的第二代单链可变区片段（scFvs），使其具有更广泛的进化枝特异性。在初步结果中，我们以迄今为止报道的任何人源化抗体的最高分辨率（1.55 E）解析了KD-247的未配体抗原结合片段（Fab）的晶体结构。我们还获得了基于野生型KD-247的克隆的scFv，用于构建突变体，并进行了分子建模研究，这将最初指导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：1。为了确定KD-247有效中和广谱进化枝B分离株的原因2.设计、构建和评估有效结合进化枝B和非进化枝B分离株的scFv KD-247。实现本提案的目的将提供结构、生物化学和病毒学知识，这些知识将指导具有改善的效力和更广泛的中和能力的第二代抗V3抗体的设计和工程化。此外，我们预期的高分辨率结构还将提供有关分子蛋白质-蛋白质接触的重要信息，这些信息对于抗体识别特定抗原至关重要。基于结构的抗HIV mAb工程是一种创新方法，可以作为设计其他治疗方法的范例。 公共卫生相关性：该项目将确定KD-247的晶体结构，KD-247是一种目前正在临床试验中的单克隆抗体，这将有助于我们理解为什么它对许多但不是所有类型的HIV都有效。这些知识将帮助我们设计具有更广泛中和能力的第二代抗体。

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.