Single-Domain Protein A-Engineered Magnetic Nanoparticles: Toward a Universal Strategy to Site-Specific Labeling of Antibodies for Targeted Detection of Tumor Cells

Abstract

Highly monodisperse magnetite nanocrystals (MNC) were synthesized in organic media and transferred to the water phase by ultrasound-assisted ligand exchange with an iminodiacetic phosphonate. The resulting biocompatible magnetic nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, and magnetorelaxometry, indicating that this method allowed us to obtain stable particle dispersions with narrow size distribution and unusually high magnetic resonance T(2) contrast power. These nanoparticles were conjugated to a newly designed recombinant monodomain protein A variant, which exhibited a convincingly strong affinity for human and rabbit IgG molecules. Owing to the nature of antibody-protein A binding, tight antibody immobilization occurred through the Fc fragment thus taking full advantage of the targeting potential of bound IgGs. If necessary, monoclonal antibodies could be removed under controlled conditions regenerating the original IgG-conjugatable MNC. As a proof of concept of the utility of our paramagnetic labeling system of human IgGs for biomedical applications, anti-HER-2 monoclonal antibody trastuzumab was immobilized on hybrid MNC (TMNC). TMNC were assessed by immunoprecipitation assay and confocal microscopy effected on HER-2-overexpressing MCF-7 breast cancer cells, demonstrating excellent recognition capability and selectivity for the target membrane receptor.

Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning

“…Conjugation of the peptide to a dye or MRI contrast agent could be used for in vivo (tumor) imaging in patients undergoing antibody treatment. Non-covalent antibody conjugates have been explored by several groups (41)(42)(43), mostly making use of generic Fc-binding protein domains (e.g. Staphylococcus aureus protein A).…”

Gelatinase activity imaged by activatable cell-penetrating peptides in cell-based and in vivo models of stroke

“…40 Targeted MRI contrast agents are designed to localize to specific cell types or tissues through active binding mechanisms, which may exploit the conjugation of the signal enhancer with suitable markers. 41 With target-specific MR contrast agents, MRI can elucidate molecular mechanisms responsible for disease progression. 42 To increase the sensitivity of MRI for detecting low abundance molecules, one approach is to utilize molecular amplification -in our case using dendrimers incorporating many molecules of Gd, as well as a few fluorophores.…”

Immobilization of carboxypeptidase from Sulfolobus solfataricuson magnetic nanoparticles improves enzyme stability and functionality in organic media

“…In our ongoing effort in designing innovative approaches for the orientation-controlled conjugation of proteins on metal-based nanoparticles for biotechnological and biomedical applications [5,27-31], we have recently investigated the modulation of the structural properties of the immobilized protein from a biophysical point of view [13]. In the present paper, we report the results of a study conducted on a hyperthermophilic enzyme, namely CP Sso , aimed at determining the effect of its immobilization on superparamagnetic nanoparticles on the biological activity of the enzyme.…”