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Antibody Fragments Humanization: Beginning with the End in Mind

  • Nicolas Aubrey
  • Philippe Billiald
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1904)

Abstract

Molecular engineering has made possible to reformat monoclonal antibodies into smaller antigen-binding structures like scFvs, diabodies, Fabs with new potential in vivo applications because they do not induce Fc-mediated functions. However, most of these molecules are from rodent origin. As a consequence, they are immunogenic and approval for administration to humans requires prior humanization. Today, there is no well-identified strategy to create recombinant humanized antibody V-domains that preserve the antigen-binding characteristics of the parental antibody associated with high stability and solubility. Here, we propose a strategy that consists in grafting CDRs onto properly chosen human antibody frameworks in order to reduce immunogenicity. A flowchart indicates the way to proceed in order to introduce an internal affinity purification tag while structural refinements are proposed to maintain antigen-binding characteristics. The best humanized candidates are identified through selection steps including in silico analysis, research scale production followed by early functional evaluation, purification assays, aggregation, and stability assessment.

Key words

Monoclonal antibody Therapeutic antibody Fab scFv Protein L Humanization CDR grafting 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.UMR Université-INRA ISP 1282, BioMAP, Université de ToursToursFrance
  2. 2.School of PharmacyParis-Sud UniversityChâtenay-MalabryFrance

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