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

Aligarh Muslim University, India
PLoS ONE (Impact Factor: 3.23). 08/2012; 7(8):e43077. DOI: 10.1371/journal.pone.0043077
Source: PubMed


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, Z(EGFR) and Z(HER2), 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 Z(EGFR) and Z(HER2) 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..

Download full-text


Available from: Dongwook Kim
  • [Show abstract] [Hide abstract]
    ABSTRACT: The C-terminal coiled-coil region of mouse and human cartilage matrix protein (CMP) self-assembles into a parallel trimeric complex. Here, we report a general strategy for the development of highly stable trimeric targeting ligands (tribody), against epidermal growth factor receptor (EGFR) and prostate-membrane specific antigen (PSMA) as examples, by fusing a specific target-binding moiety with a trimerization domain derived from CMP. The resulting fusion proteins can efficiently self-assemble into a well-defined parallel homotrimer with high stability. Surface plasmon resonance (SPR) analysis of the trimeric targeting ligands demonstrated significantly enhanced target binding strength compared with the corresponding monomers. Cellular binding studies confirmed that the trimeric targeting ligands have superior binding strength towards their respective receptors. Significantly, EGFR-binding tribody was considerably accumulated in tumor in xenograft mice bearing EGFR positive tumors, indicating its effective cancer targeting feature under in vivo conditions. Our results demonstrate that CMP-based self-assembly of tribody can be a general strategy for the facile and robust generation of trivalent targeting ligands for a wide variety of in vitro and in vivo applications.
    No preview · Article · Sep 2013 · Biochemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: The polygenetic nature of most cancers emphasizes the necessity of cancer therapies that target multiple essential signaling pathways. However, there is a significant paucity of targeting ligands with multi-specificities for targeted delivery of biomaterials. To address this unmet need, we generated a tetraspecific targeting ligand that recognizes four different cancer biomarkers, including VEGFR2, αvβ3 integrin, EGFR, and HER2 receptors, which have been implicated in numerous malignant tumors. The tetraspecific targeting ligand was constructed by sequentially connecting four targeting ligand subunits via flexible linkers, yielding a fusion protein that can be highly expressed in Escherichia coli and readily purified to near homogeneity. Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI) studies and extensive cellular binding analyses indicated that all the targeting ligand subunits in the tetraspecific fusion protein recognized their target receptors proximately to the corresponding monospecific ligands. The resulting tetraspecific targeting ligand was applied for the delivery of nanomaterials such as gold nanoparticles (AuNPs) for targeted hyperthermic killing of various cancer cell lines with biomarkers of interest expressed. We demonstrate that the tetraspecific ligand can be facilely introduced on the surface of AuNPs and efficient target-dependent killing of cancer cells can be achieved only when the AuNPs are conjugated with the tetraspecific ligand. Significantly, the tetraspecific ligand simultaneously interacts with more than one receptors, such as EGFR and HER2 receptors, when they are expressed on the surface of the same cell, as demonstrated by in vitro binding assays and cell binding analyses. Our results demonstrate that the tetraspecific ligand, through multivalency and synergistic binding, can be readily used to generate various ‘smart’ biomaterials with greatly broadened tumor targeting range for simultaneous targeting of multiple signaling pathways on many different cancer types.
    No preview · Article · Jul 2014 · Biomaterials
  • [Show abstract] [Hide abstract]
    ABSTRACT: The scientific review surveys release mechanisms as classified on the basis of linker type for various drugs conjugated to nanocarriers and discusses how such mechanisms are designed and incorporated specifically into the delivery platform to achieve specific targeted release in diseased cells. Nanotechnology for drug delivery provides a diverse array of delivery platforms, and is uniquely suited for improving the safety and efficacy profiles of current chemotherapeutic agents. Nanoscale entities are available in various shapes such as globular particles, tubes, and rods, and can serve as versatile nanocarrier platforms that have the potential to provide multifunctional properties.
    No preview · Article · Apr 2015 · Chemical Reviews