title: Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.
abstract: A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.
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: Niklas
authors.initials: N
authors.lastname: Weber
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: Protein Engineering and Antibody Technologies, Merck Serono, Merck KGaA, Frankfurter Straße 250, D-64293 Darmstadt, Germany.
authors.forename: Achim
authors.initials: A
authors.lastname: Doerner
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Christmann
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Christmann
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Uth
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany.
authors.forename: Janine
authors.initials: J
authors.lastname: Fritz
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Schäfer
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Steinmann
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Department Drug Design and Optimization, Saarland University, Campus C2.3, D-66123 Saarbrücken, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Empting
authors.affiliation: Goethe-University Frankfurt, Faculty of Biosciences, Max-von-Laue-Str. 13, D-60438 Frankfurt am Main, Germany; University Hospital Frankfurt, Department of Anesthesiology, Intensive-Care Medicine and Pain Therapy, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
authors.forename: Pia
authors.initials: P
authors.lastname: Ockelmann
authors.affiliation: Clinic for Birds, Reptiles, Amphibians and Fish, Justus-Liebig University, Gießen, Frankfurter Str. 91-93, D-35392 Giessen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Lierz
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany. Electronic address: Kolmar@Biochemie-TUD.de.
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000276:immunology*; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000276:immunology*; D000071858:Epithelial Cell Adhesion Molecule; D000074289:High-Temperature Requirement A Serine Peptidase 1; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D066298:In Vitro Techniques; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D036104:Receptor, EphA2 / Q000737:chemistry* / Q000276:immunology; D011946:Receptors, Antigen / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000276:immunology; D012754:Sharks / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D001324:Autoantigens; C063762:CDR1 protein, human; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D007136:Immunoglobulins; D009419:Nerve Tissue Proteins; D019151:Peptide Library; D011946:Receptors, Antigen; C092388:nurse shark antigen receptor; D036104:Receptor, EphA2; D000074289:High-Temperature Requirement A Serine Peptidase 1; C474012:HTRA1 protein, human; D012697:Serine Endopeptidases
keywords: Semi-synthetic library; Shark vNAR antibody; Stepwise in vitro affinity maturation; Yeast surface display