Alicanto: Proteogenomic discovery of single chain antibodies in llama

Alicanto：美洲驼单链抗体的蛋白质组学发现

基本信息

批准号：
10011252
负责人：
Stefano Romoli Bonissone
金额：
$ 60.46万
依托单位：
DIGITAL PROTEOMICS LLC
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-20 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10011252
关键词：
Active Sites Affinity Alanine Animals Antibodies Antibody Response Antigen Targeting Antigens B-Lymphocytes Bacteriophages Behavior Binding Categories Cations Cities Cloning Computer Analysis Computer software Computing Methodologies Crystallography Custom Cytometry Data Databases Diagnostic Engineering Ensure Enzyme-Linked Immunosorbent Assay Enzymes Epitopes Escherichia coli Fishes Gene Conversion Genes Genetic Goals Histology Image Immune response Immune system Immunization Immunoprecipitation Individual Keyhole Limpet Hemocyanin Libraries Light Link Llama Mass Spectrum Analysis Methods Modality Molecular Conformation Organism Peptides Performance Pesticides Phage Display Phase Phylogenetic Analysis Process Proteins RNA RNA Editing Reagent Research Sampling Biases Serum Site Structure Technology Temperature Therapeutic TimeLine Transcript Western Blotting Work antigen binding base candidate selection commercial application comparative head-to-head comparison immunological diversity in silico interest lead candidate next generation sequencing novel polypeptide proteogenomics response screening small molecule structural biology synthetic construct tandem mass spectrometry

项目摘要

Heavy chain-only antibodies (HCAbs) are a unique class of antibodies only produced in camelids and cartilagenous fish. Unlike conventional antibodies that consist of two heavy chains and two light chains, HCAbs consist only of heavy chains. The antigen-binding portion of the camelid HCAb, called the VHH, is a polypeptide fragment with wide utility across crystallography, imaging, and therapeutics. The small size and comparatively simple structure of the VHH as compared to conventional antibodies makes them economical to produce and structurally stable at a wider pH and temperature range. Growing evidence suggests that the epitopes targeted by HCAbs are distinct from those targeted by conventional antibodies. Concave domains, such as enzyme active sites, are one category that are preferentially targeted by HCAbs than conventional antibodies. This is in part due to the ability of VHHs to produce convex conformations which enables targeting of sites that are inaccessible to conventional antibodies. Phage display is the predominant method for discovering novel VHHs. HCAb transcripts are cloned into phagemids, and phages that express a VHH that binds the target are enriched through the process of panning. Attrition at cloning, panning, or final selection reduces the accessible diversity of the immune system, and delivers antibodies that may not be as diverse as the response mounted by the original host organism. In contrast to display-based methods, Alicanto combines next-generation sequencing and mass spectrometry to directly identify antigen-specific circulating HCAbs from camelids. Assessment of circulating HCAbs in serum is the primary method by which an immunization is determined to be successful. While the serum is discarded after screening in the phage display workflow, Alicanto uses mass spectrometry to identify the individual HCAbs comprising the target-specific circulating antibodies. This enables Alicanto to discover low abundance antibodies against challenging targets such as peptides and small molecules. By constructing an in silico library of HCAb sequences using next-generation sequencing, Alicanto precludes the need to clone into an intermediate host such as phages. Through direct analysis of the immune response of llamas, Alicanto will deliver more, high affinity VHHs for use in research, diagnostics, and therapeutics.

仅重链抗体 (HCAb) 是一类独特的抗体，仅在骆驼科动物和软骨鱼。与由两个重链组成的传统抗体不同链和两条轻链，HCAb 仅由重链组成。抗原结合部分骆驼科 HCAb，称为 VHH，是一种具有广泛用途的多肽片段晶体学、成像和治疗学。体积小，结构相对简单与传统抗体相比，VHH 的优点使其生产和生产更加经济在更宽的 pH 值和温度范围内结构稳定。越来越多的证据表明 HCAb 靶向的表位不同于那些被常规抗体靶向。凹结构域，例如酶活性位点，是其中之一与传统抗体相比，HCAb 优先靶向的类别。这是部分由于 VHH 能够产生凸形构象，从而能够靶向位点这是传统抗体无法达到的。噬菌体展示是发现新型 VHH 的主要方法。 HCAb 转录本被克隆到噬菌粒中，表达结合靶标的 VHH 的噬菌体被富集通过平移的过程。克隆、淘选或最终选择时的损耗会降低免疫系统的多样性，并提供可能不那么多样化的抗体作为原始宿主生物体的反应。与基于显示的方法相比，Alicanto 结合了下一代测序和质谱法直接鉴定来自骆驼科动物的抗原特异性循环 HCAb。评估血清中的循环 HCAb 是免疫接种的主要方法决心要成功。噬菌体展示筛选后血清被丢弃工作流程中，Alicanto 使用质谱法来识别组成的单个 HCAb 目标特异性循环抗体。这使得阿利坎托能够发现低丰度针对具有挑战性的靶标（例如肽和小分子）的抗体。通过构建使用下一代测序技术的 HCAb 序列计算机文库，Alicanto 排除了需要克隆到中间宿主，例如噬菌体。通过直接免疫分析根据美洲驼的反应，Alicanto 将提供更多、高亲和力的 VHH 用于研究，诊断和治疗。