Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two

Abstract

The tenth human fibronectin type three domain ((10)Fn3) is a small (10 kDa), extremely stable and soluble protein with an immunoglobulin-like fold, but without cysteine residues. Selections from (10)Fn3-based libraries of proteins with randomized loops have yielded high-affinity, target-specific antibody mimics. However, little is known about the biophysical properties of such antibody mimics, which will determine their suitability for in vitro and medical applications. We characterized target binding and biophysical properties of two related (10)Fn3-based antibody mimics that bind vascular endothelial growth factor receptor two (VEGF-R2). The first antibody mimic, which has a dissociation constant (K(d)) of 13 nM, is highly stable [melting temperature (T(m))=62 degrees C] and soluble, whereas the second, which binds VEGF-R2 with 40 x higher affinity, is less stable (T(m) < 40 degrees C) and relatively insoluble. We used our understanding of these two (10)Fn3 derivatives and of wild-type (10)Fn3 structure to engineer the next generation of antibody mimics, which have an improved combination of high affinity (K(d)=0.59 nM), stability (T(m)=53 degrees C) and solubility. Our findings illustrate that (10)Fn3-based antibody mimics can be engineered for favorable biophysical properties even when 20% of the wild-type (10)Fn3 sequence is mutated in order to satisfy target-binding requirements.

Picomolar Affinity Fibronectin Domains Engineered Utilizing Loop Length Diversity, Recursive Mutagenesis, and Loop Shuffling

“…The 10th type III domain of human fibronectin (Fn3) has been demonstrated as an effective scaffold for molecular recognition.1 -9 It is a small (10 kDa), stable (T m =84 °C) 7 cysteine-free beta-sandwich protein that maintains its native fold in reducing environments and can be produced at up to 50 mg/L in Escherichia coli. 9 The protein fold resembles an immunoglobulin variable domain and contains three solvent-exposed loops, termed BC, DE, and FG, for the β strands they connect, which are structurally analogous to antibody complementaritydetermining regions (CDRs).…”

Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity

“…Over 800 unique sequences were identified; however, many of these clones exhibited some relatedness to each other and thus could be grouped into a few dozen families. Cloned DNA sequences were expressed in Escherichia coli, and proteins were partially purified using a high-throughput immobilizedmetal ion affinity chromatography (IMAC) method similar to that described previously (42). The partially purified Adnectins were first assessed by size exclusion chromatography (SEC), and proteins that exhibited Յ50% aggregation progressed to an enzymelinked immunosorbent assay (ELISA) that detected binding of the molecules to the CD4-Fc target.…”

Pharmacologic Profile of the Adnectin BMS-962476, a Small Protein Biologic Alternative to PCSK9 Antibodies for Low-Density Lipoprotein Lowering

“…Generation of the Adnectins followed procedures described previously (Parker et al, 2005;Lipovsek, 2011). Briefly, an Adnectin DNA library of high diversity (2.3 Â 10 12 ) produced by randomizing the 3 variable loops (denoted BC, DE, and FG) of 10Fn3 was taken through multiple rounds of mRNA display.…”

“…Adnectins are based on the 10th-type III-domain (10Fn3) of human fibronectin, whose variable loops can be efficiently engineered to introduce surfaces that bind therapeutic targets with high affinity and specificity (Koide et al, 1998;Wojcik et al, 2010). Adnectins are small (#12 kDa), compact proteins without sequence homology to immunoglobulins but possessing a b-sheet fold structure with diversified loops analogous to antibody variable regions (Parker et al, 2005;Lipovsek, 2011). Adnectins have no disulfides and are not glycosylated, exhibit high thermal stability and monomeric solution behavior, and are efficiently produced using bacterial expression systems.…”