Alicanto: Proteogenomic discovery of single chain antibodies in llama

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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.