Alicanto: Proteogenomic discovery of single chain antibodies in llama

仅重链抗体(HCAbs)是一类独特的抗体，仅在 骆驼和软骨鱼。与传统的由两个重的抗体组成的抗体不同 HCAb有两条轻链和两条重链，仅由重链组成。病毒的抗原结合部分 骆驼HCAb被称为VHH，是一种具有广泛用途的多肽片段 结晶学、成像学和治疗学。体积小，结构相对简单 VHH与传统抗体相比，使它们的生产和 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所针对的表位与那些 以常规抗体为靶点。凹陷结构域，如酶活性部位，是一种 与传统抗体相比，HCAb优先靶向的类别。这在一定程度上 由于VHH能够产生凸形构象，从而能够靶向定位 这是常规抗体所不能达到的。 噬菌体展示是发现新型VHH的主要方法。HCAb转录本 被克隆到噬菌体中，表达与靶标结合的VHH的噬菌体得到丰富 通过平底锅的过程。在克隆、平移或最终选择时的损耗减少了 可获得的免疫系统多样性，并提供可能不那么多样化的抗体 作为原始寄主生物体的反应。 与基于显示的方法不同，Alicanto结合了下一代测序 和质谱学直接从骆驼中鉴定抗原特异性循环HCAb。 评估血清中循环HCAb是免疫接种的主要方法 决心要成功。而血清在噬菌体展示中筛选后被丢弃 工作流程，Alicanto使用质谱学来识别组成 靶标特异性循环抗体。这使得Alicanto能够发现低丰度 针对具有挑战性的目标的抗体，如多肽和小分子。通过构建 使用下一代测序的HCAb序列的电子文库中，Alicanto排除了 需要克隆到中间宿主，如噬菌体。通过对免疫系统的直接分析 大羊驼的反应，Alicanto将提供更多、高亲和力的VHH用于研究， 诊断学和治疗学。

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.