Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

Abstract

Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2, were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric ZEGFR and ZHER2 ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents..

Tetraspecific ligand for tumor-targeted delivery of nanomaterials

“…The coding sequences for VEGFR2 binding FN3 VEGFR2 and α v β 3 binding FN3 α v β 3 domains were synthesized by GenScript (Piscataway, NJ). The coding sequences for EGFR-binding Z EGFR and HER2-binding Z HER2 domains were amplified from the genes coding heptameric ligands as we recently reported [17]. The amplified coding sequences were assembled to form a fusion gene by PCR using primers described in Supplemental Table 1.…”

“…The quality of the purified proteins was checked with SDS-PAGE. The labeling of purified proteins by fluorescein isothiocyanate (FITC) (ACROS organics, Geels, Belgium) and Alexa Fluor 555 carboxylic acid, succinimidyl ester (Alexa 555) (Life technologies, Grand Island, NY) was performed according to the procedures as we published previously [17]. …”

Targeted Delivery of 111In Into the Nuclei of EGFR Overexpressing Cells via Modular Nanotransporters With Anti-EGFR Affibody

“…, 2 0 0 7 ) l e a d s t o s t i m u l a t i o n o f proliferation, inhibit apoptosis, and promote migration (Seshacharyulu et al, 2012). In order to avoid ligand-mediated activation of the downstream cascades researchers developed alternative EGFR ligands such as EGFt, a truncated form of human EGF (hEGF) lacking the eight C-terminal amino acids (Panosa et al, 2015), artificial EGFR ligands such as small antibody-like protein on the basis of Z-domain of A-protein: anti-EGFR affibody (Z EGFR :1907) (Kim et al, 2012;Stahl et al, 2017), and anti-EGFR nanobody (Roovers et al, 2007).…”

Tribody: Robust Self-Assembled Trimeric Targeting Ligands with High Stability and Significantly Improved Target-Binding Strength

“…2–5 The protein domains capable of forming multimeric complex have been extensively investigated to generate recombinant proteins to achieve avidity effect through multivalency. 1,6 To develop a robust system that allows for facile generation of targeting ligands with multivalent features, the multimerization domains should be of small sizes and possess favorable biophysical properties, including thermal stability, resistance to protease, and cost effectiveness in its production, while still able to generate highly stable multimeric complex that can display multiple target-binding moieties in parallel. Different scaffolds that allow for enhanced avidity have been reported.…”

“…1,2,5–7 These scaffolds include the bacteriophage T4 foldon domain, collagen like peptide (Gly-Pro-Pro) 10 , NC1 domain of collagen XV and XVIII domain, and GCN leucine zipper domain for trimers, 1,2,8,9,10 streptavidin and transcription factor p53 for tetramers, 11 the B-subunit of bacterial verotoxin and cartilage oligomeric matrix protein (COMP) for pentamers, 12,13 and recently the hyperthermophilic Sm protein for heptamers. 6 However, most of these scaffolds are derived from non-human proteins and have limited clinical application due to immunogenicity. Ideally, the multimerization domain should be a highly conserved extracellular protein that is abundant in mouse and human proteomes, which could result in less immunogenicity and allow for smooth transition from animal studies to translational and clinical investigations.…”