title: SNAP-Tag Technology: A Useful Tool To Determine Affinity Constants and Other Functional Parameters of Novel Antibody Fragments.
abstract: Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore, we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore, we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry, and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME , 52074 Aachen, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Niesen
authors.affiliation: Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University , 52074 Aachen, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Sack
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME , 52074 Aachen, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Seidel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME , 52074 Aachen, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Fendel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME , 52074 Aachen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME , 52074 Aachen, Germany.|Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University , 52074 Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME , 52074 Aachen, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Stein
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D000077544:Panitumumab; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D057127:Single-Chain Antibodies; D000077544:Panitumumab; D066246:ErbB Receptors