A novel epitope-presenting thermostable scaffold for the development of highly specific insulin-like growth factor-1/2 antibodies

Abstract

High sequence and structural homology between mature human insulin-like growth factors IGF-1 and IGF-2 makes serological discrimination by immunodiagnostic IGF tests a challenging task. There is an urgent need for highly specific IGF-1 and IGF-2 antibodies, yet only a short sequence element, i.e. the IGF loop, provides enough difference in sequence to discriminate between the two molecules. We sought to address this unmet demand by investigating novel chimeric immunogens as carriers for recombinant peptide motif grafting. We found Thermus thermophilus sensitive to lysis D (SlyD) and Thermococcus gammatolerans SlyD FK-506–binding protein (FKBP) domains suitable for presentation of the predefined epitopes, namely the IGF-1 and IGF-2 loops. Chimeric SlyD-IGF proteins allowed for the development of exceptionally specific IGF-1 and IGF-2 monoclonal antibodies. The selected antibodies bound with high affinity to the distinct IGF epitopes displayed on the protein scaffolds, as well as on the mature human IGF isoforms. The respective SlyD scaffolds display favorable engineering properties in that they are small, monomeric, and cysteine-free and can be produced in high yields in a prokaryotic host, such as Escherichia coli. In conclusion, FKBP domains from thermostable SlyD proteins are highly suitable as a generic scaffold platform for epitope grafting.

Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies

“…Hydrophobic proteins have been employed for biopanning either as truncated soluble recombinant extracellular domains (ECDs) or reconstituted in an artificial lipid environment such as liposomes [ 5 ] or nanodiscs [ 6 ]. Other approaches to present antigens from hydrophobic regions include the introduction of stabilizing mutations typically focused on transmembrane helices [ 7 ], the use of purified target-enriched cell membranes [ 8 ], and the use of scaffold proteins to present grafted native epitopes [ 9 ]. Recombinant ECDs may not be ideal for the isolation of target-specific antibodies due to the potential for non-native folding and the removal or inclusion of epitopes resulting from the truncation process or epitopes previously inaccessible in the membrane-bound state.…”

Treadmill training improves cognitive function by increasing IGF2 targeted downregulation of miRNA‐483

“…The insulin-like growth factor (IGF) axis plays an essential role in normal growth and development. 6 IGF2 is now known as a multifunctional growth regulator in the insulin-like growth family that promotes cell differentiation and proliferation and is associated with the regulation of physical activity. 7 Increasing evidence has demonstrated the functional significance of IGF2 in hippocampal-dependent learning and memory of rats.…”

Treadmill Training Improves Cognitive Function via Increasing IGF2 Targeted Downregulation of miRNA-483

“…The insulin like growth factor (IGF) axis plays an essential role in normal growth and development [6].…”