Combinatorial Design of an Anticalin Directed against the Extra-Domain B for the Specific Targeting of Oncofetal Fibronectin

Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties

“…Two of them, a Fynomer® and an anticalin, are scaffold-based monomeric binding proteins obtained after primary phage display selection with binding affinities toward ED-B target in the low to medium nanomolar range (28, 29). A third protein, L19, is an scFv representing the most advanced ED-B-binding protein obtained after affinity maturation.…”

Biobetters

“…The same L19 scFv moiety is also under clinical investigation in different formats and/or combination therapies, for example as TNFα fusion protein, as well as for radio-immunotherapy (RIT). While initially L19 was described as a high-affi nity scFv having a remarkable K D in the low picomolar range (Pini et al 1998 ), a signifi cantly diminished potency was measured when reformatted to a Fab (Gebauer et al 2013 ). Indeed, detailed inspection of the scFv revealed its propensity to form a stable homodimer, and this form showed much enhanced ED-B binding activity (most likely due to an avidity effect) in a head to head comparison with both the isolated monomeric scFv and the inherently monovalent Fab.…”

“…This pertains to choosing the most suitable set of residues to be targeted for mutagenesis, whereby the total number of randomized positions is generally limited according to theoretical considerations (Richter et al 2014 ). In case of the Lcn2 scaffold it was nicely demonstrated that the library design can be optimized in a few iterative cycles, also taking advantage of X-ray structural information (Gebauer et al 2013 ). The third generation Lcn2-based Anticalin library resulting from this endeavor is highly potent in yielding binding proteins against a wide range of biomolecular targets, showing high affi nities in the nM to pM range directly after phage display selection from the naive library (Richter et al 2014 ) while still offering the potential for further improvement via in vitro affi nity maturation.…”

“…Furthermore, this Anticalin has shown potential for the immunotherapy of cancer in preclinical studies (Pieris, http://www.pieris-ag.com ). Another protein-specifi c Anticalin recognizes a disease-specifi c splice variant of the extracellular matrix protein fi bronectin (Fn) exhibiting the extra-domain B (ED-B) (Gebauer et al 2013 ). Since in adults ED-B-positive, so-called oncofetal Fn is almost exclusively expressed during tumor angiogenesis, apart from wound healing and some other pathophysiological states that involve neovascularization, it has emerged as a promising marker for various cancers and constitutes a validated target for in vivo imaging (Kaspar et al 2006 ), as already mentioned further above.…”

Neuprogrammierung von humanem Siderocalin zur Neutralisierung von Petrobactin, dem essenziellen Eisenfänger des Milzbrand‐Bazillus

“…Abbildung S1) mit picomolarer Affinitätbinden. [15] Die Prozedur durchlief verschiedene Stadien nach anfänglicher Selektion aus der Zufallsbibliothek mittels Phagen-Display.E sf olgte eine Affinitätsmaturierung durch partielle Mutagenese der zentralen fürL cn2 kodierenden Region in Kombination mit Selektion durch Phagen-Display oder bakterielles Oberflä-chen-Display sowie einem Kolonie-Screening-Assay,d er die Visualisierung von Bindungsaktivitätf ürB iotin-PB·Fe III auf Ebene einzelner Klone ermçglichte (zur detaillierten Beschreibung siehe die Hintergrundinformationen). Zu diesem Zweck haben wir sowohl PB (Abbildung 1) als auch ein biotinyliertes Derivat (Biotin-PB) nach publizierten Vorschriften synthetisiert.…”