title: Selection and in vitro characterization of human CD44v6-binding antibody fragments.
abstract: The cluster of differentiation (CD) 44v6 antigen has been suggested to be involved in tumor formation, invasion, and metastasis formation, and has been observed in a majority of primary and metastatic squamous cell carcinomas of the head and neck. Probes specifically binding to this region may be utilized as tools for the challenging tasks of early detection and targeted treatments of small residual disease. In this project, an epitope-guided phage display selection of human fragment antigen-binding (Fab) fragments with affinity to the v6 sequence was performed. A selected set of Fab fragments was shown to specifically recognize increasingly complex forms of the target sequence, both in the form of a short synthetic or recombinant peptide and in the context of a purified extracellular domain of CD44. The binding was independent of known v6-sequence variation and posttranslational modifications that are common in the CD44 protein family. Furthermore, real-time interaction measurements on antibody fragments labeled with ¹²⁵I showed specific and high-affinity binding to the antigen present on cultured head and neck squamous cell carcinoma cells. There was no cross-reactivity toward cells that lack the target protein. As hypothesized, characterization of the interaction between Fab fragments and the targets using the mathematical tool Interaction Map revealed more heterogeneous interactions on cells than with pure proteins analyzed by surface plasmon resonance. One main candidate Fab fragment with optimal affinity for all forms of the target sequence was identified. The flexible recombinant source of the Fab fragments might aid the development of tailored molecules adapted for therapeutic or diagnostic applications in the future.
authors.affiliation: Department of Proteomics, School of Biotechnology, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Nilvebrant
authors.forename: Gamze
authors.initials: G
authors.lastname: Kuku
authors.forename: Hanna
authors.initials: H
authors.lastname: Björkelund
authors.forename: Marika
authors.initials: M
authors.lastname: Nestor
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology
chemical_list: C112697:CD44v6 antigen; D018960:Hyaluronan Receptors; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D020033:Protein Isoforms