title: Isolation of a pH-Sensitive IgNAR Variable Domain from a Yeast-Displayed, Histidine-Doped Master Library.
abstract: In recent years, engineering of pH-sensitivity into antibodies as well as antibody-derived fragments has become more and more attractive for biomedical and biotechnological applications. Herein, we report the isolation of the first pH-sensitive IgNAR variable domain (vNAR), which was isolated from a yeast-displayed, semi-synthetic master library. This strategy enables the direct identification of pH-dependent binders from a histidine-enriched CDR3 library. Displayed vNAR variants contained two histidine substitutions on average at random positions in their 12-residue CDR3 loop. Upon screening of seven rounds against the proof-of-concept target EpCAM (selection for binding at pH 7.4 and decreased binding at pH 6.0), a single clone was obtained that showed specific and pH-dependent binding as characterized by yeast surface display and biolayer interferometry. Potential applications for such pH-dependent vNAR domains include their employment in tailored affinity chromatography, enabling mild elution protocols. Moreover, utilizing a master library for the isolation of pH-sensitive vNAR variants may be a generic strategy to obtain binding entities with prescribed characteristics for applications in biotechnology, diagnostics, and therapy.
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 4, D-64287, Darmstadt, Germany.
authors.forename: Doreen
authors.initials: D
authors.lastname: Könning
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 4, D-64287, Darmstadt, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Zielonka
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 4, D-64287, Darmstadt, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Sellmann
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 4, D-64287, Darmstadt, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Schröter
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 4, D-64287, Darmstadt, Germany.
authors.forename: Julius
authors.initials: J
authors.lastname: Grzeschik
authors.affiliation: Protein Engineering and Antibody Technologies, Merck-Serono, Merck KGaA, Frankfurter Straße 250, D-64293, Darmstadt, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 4, D-64287, Darmstadt, Germany. Kolmar@Biochemie-TUD.de.
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D029941:Fish Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D015723:Gene Library; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012754:Sharks; D061905:Single-Domain Antibodies / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D029941:Fish Proteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease
keywords: IgNAR; Shark antibody; Single domain antibody; Yeast surface display; pH-dependent binding; vNAR
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