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

• 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
• 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

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.

纯重链抗体（HCAb）是一类独特的抗体，仅在哺乳动物中产生。 骆驼科动物和软骨鱼类。与由两个重链组成的常规抗体不同， HCAb含有两条重链和两条轻链，HCAb仅由重链组成。的抗原结合部分 骆驼科动物HCAb，称为VHH，是一种多肽片段， 结晶学、成像学和治疗学。体积小，结构相对简单 与常规抗体相比，VHH的减少使得它们的生产经济， 在较宽的pH和温度范围内结构稳定。 越来越多的证据表明，HCAb靶向的表位与那些 常规抗体的靶向。凹域，如酶活性位点，是一种 HCAb比常规抗体优先靶向的类别。这部分是 由于VHH产生凸构象的能力， 这些是常规抗体所无法接触到的。 噬菌体展示是发现新型VHH的主要方法。HCAb转录本 克隆到噬菌粒中，并富集表达结合靶的VHH的噬菌体 通过淘洗的过程。在克隆、淘选或最终选择时， 免疫系统的多样性，并提供抗体， 作为原始宿主生物的反应。 与基于显示的方法相比，Alicanto结合了下一代测序技术， 和质谱法直接鉴定来自骆驼科动物的抗原特异性循环HCAb。 评估血清中循环HCAb是免疫接种的主要方法， 决心要成功。而在噬菌体展示中筛选后丢弃血清 工作流程中，Alicanto使用质谱法来鉴定包含HCAb的单个HCAb。 目标特异性循环抗体。这使得Alicanto能够发现低丰度 针对挑战性靶标如肽和小分子的抗体。通过构造 Alicanto利用下一代测序技术建立了HCAb序列的计算机文库， 需要克隆到一个中间宿主，如IBM。通过直接分析免疫 针对美洲驼的反应，Alicanto将提供更多高亲和力的VHH用于研究， 诊断和治疗。