title: The Shark Strikes Twice: Hypervariable Loop 2 of Shark IgNAR Antibody Variable Domains and Its Potential to Function as an Autonomous Paratope.
abstract: In this present study, we engineered hypervariable loop 2 (HV2) of the IgNAR variable domain in a way that it solely facilitates antigen binding, potentially functioning as an autonomous paratope. For this, the surface-exposed loop corresponding to HV2 was diversified and antigen-specific variable domain of IgNAR antibody (vNAR) molecules were isolated by library screening using yeast surface display (YSD) as platform technology. An epithelial cell adhesion molecule (EpCAM)-specific vNAR was used as starting material, and nine residues in HV2 were randomized. Target-specific clones comprising a new HV2-mediated paratope were isolated against cluster of differentiation 3ε (CD3ε) and human Fcγ while retaining high affinity for EpCAM. Essentially, we demonstrate that a new paratope comprising moderate affinities against a given target molecule can be engineered into the vNAR scaffold that acts independent of the original antigen-binding site, composed of complementarity-determining region 3 (CDR3) and CDR1.
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, 64287, Darmstadt, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Zielonka
authors.forename: Martin
authors.initials: M
authors.lastname: Empting
authors.forename: Doreen
authors.initials: D
authors.lastname: Könning
authors.forename: Julius
authors.initials: J
authors.lastname: Grzeschik
authors.forename: Simon
authors.initials: S
authors.lastname: Krah
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.forename: Stephan
authors.initials: S
authors.lastname: Dickgießer
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001666:Binding Sites, Antibody / Q000235:genetics; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012754:Sharks / Q000235:genetics* / Q000276:immunology*
chemical_list: D007143:Immunoglobulin Heavy Chains