Increasing thermal stability and improving biodistribution of VEGFR2-binding affibody molecules by a combination of in silico and directed evolution approaches

Abstract

The family of vascular endothelial growth factor (VEGF) ligands and their interactions with VEGF receptors (VEGFRs) play important roles in both pathological and physiological angiogenesis. Hence, agonistic and antagonistic ligands targeting this signaling pathway have potential for both studies on fundamental biology and for development of therapies and diagnostics. Here, we engineer VEGFR2-binding affibody molecules for increased thermostability, refolding and improved biodistribution. We designed libraries based on the original monomeric binders with the intention of reducing hydrophobicity, while retaining high affinity for VEGFR2. Libraries were displayed on bacteria and binders were isolated by fluorescence-activated cell sorting (FACS). In parallel, we used an automated sequence- and structure-based in silico algorithm to identify potentially stabilizing mutations. Monomeric variants isolated from the screening and the in silico approach, respectively, were characterized by circular dichroism spectroscopy and biosensor assays. The most promising mutations were combined into new monomeric constructs which were finally fused into a dimeric construct, resulting in a 15 °C increase in melting temperature, complete refolding capability after heat-induced denaturation, retained low picomolar affinity and improved biodistribution profile in an in vivo mouse model. These VEGFR2-binding affibody molecules show promise as candidates for further in vivo studies to assess their suitability as molecular imaging and therapeutic agents.

Material-specific binding peptides empower sustainable innovations in plant health, biocatalysis, medicine and microplastic quantification

“…405 Mutations introduced to enhance affinity for target compounds can impact the structural integrity or stability of affibodies in various environmental conditions. 406 Therefore, diverse protein engineering strategies were proposed to achieve high-affinity affibodies without compromising structural stability including directed evolution, the protein repair one-stop shop (PROSS) in silico algorithm, 407 gradient sitewise diversity generation and screening, 408 and the introduction of intramolecular disulphide bonds. 409 In recent decades, affibodies for over 40 targets, including amyloid b peptide, epidermal growth factor receptor, fibrinogen, interleukins, tumour necrosis factor, or insulin, were already generated for the applications in biomedicine for both in vitro (bioimaging, diagnostics) and in vivo (therapeutics) use.…”

GMMA can stabilize proteins across different functional constraints

“…Many algorithms, most notably PROSS [9,10], sample only from a set of substitutions commonly seen throughout evolution. Such approaches have been successful for stabilizing many classes of proteins, including enzymes, transporters, and binding proteins [11][12][13]. Still, identifying positions not to mutate and sets of substitutions that do not disrupt function is often performed empirically or with heuristics that may not transfer across to other systems.…”

Moderate‐Affinity Affibodies Modulate the Delivery and Bioactivity of Bone Morphogenetic Protein‐2

“…[42,43] Affibodies are currently being tested clinically and preclinically as targeting agents for HER2 + breast cancer cells, [44,45] and for the detection of other biological markers, such as CD69 cell markers for early detection of activated immune cells [46] and vascular endothelial growth factor receptor-2 (VEGFR2) expression for analyzing angiogenesis signaling pathways. [47] Moreover, affibodies have also recently been used to tune the release of fibroblast growth factor-2 (FGF-2), [40] insulin-like growth factor-1 (IGF-1), and pigment epitheliumderived factor (PEDF). [39] Their clinical benefit is derived from their relatively stable structure under physiological conditions, the diversity of proteins to which they can bind, and the ability to modify their binding affinity by changing 13 to 17 amino acids at the binding interface between the affibody and target protein.…”