In Vivo Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures

Abstract

Chemically Self-Assembled Nanorings (CSANs) are made of dihydrofolate reductase (DHFR) fusion proteins and have been successfully used in vitro for cellular cargo delivery and cell surface engineering applications. However, CSANs have yet to be evaluated for their in vivo stability, circulation and tissue distribution. In an effort to evaluate CSANs in vivo, we engineered a site-specifically PEGylated epidermal growth factor receptor (EGFR) targeting DHFR molecules, characterized their self-assembly in to CSANs with bivalent methotrexates (bis-MTX), visualized their in vivo tissue localization by microPET/CT imaging and determined their ex vivo organ biodistribution by tissue-based gamma counting. A dimeric DHFR (DHFR2) molecule fused with a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate a site-specific ketone functionality using unnatural amino-acid mutagenesis. Aminooxy-PEG, of differing chain lengths, was successfully conjugated to the protein using oxime chemistry. These proteins were self-assembled into CSANs with bis-MTX DHFR dimerizers and characterized by size exclusion chromatography and dynamic light scattering. In vitro binding studies were performed with fluorescent CSANs assembled using bis-MTX-FITC, while in vivo microPET/CT imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTA[64Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by mouse macrophages (RAW 264.7) up to 40% without altering the CSAN’s binding affinity towards U-87 MG glioblastoma cells in vitro. A significant time dependent tumor accumulation of 64Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and biodistribution studies in mice bearing U-87 MG xenografts. PEGylated CSANs demonstrated a reduced uptake by the liver, kidneys and spleen resulting in high contrast tumor imaging within an hour of intravenous injection (9.6% ID/g), and continued to increase up to 24 h (11.7% ID/g) while the background signal diminished. CSANs displayed an in vivo profile between that of rapidly clearing small molecules and slow clearing antibodies. Thus CSANs offer a modular, programmable and stable protein based platform that can be used for in vivo drug delivery and imaging applications.

Eradication of Established Tumors by Chemically Self-Assembled Nanoring Labeled T Cells

“…To characterize the in vivo biodistribution of PAR therapy we generated radiolabeled αEpCAM/αCD3 bispecific CSANs and αCD3 non-targeted CSANs based on our previously developed methodology. 30 Monomeric DHFR 2 -αEpCAM and DHFR 2 -αCD3 were incubated with three equivalents of bis-MTX-DOTA[ 64 Cu] at room temperature to form the relevant 64 Cu labeled CSAN constructs as previously described, followed by purification with a Bio-Spin6 SEC column resulting in labelling efficiencies of greater than 95% for all constructs. NSG mice (n = 4) bearing orthotopic MCF-7-Luc tumors were engrafted with 20 million PBMCs by IV infusion followed 4 days later by IV injection of 180 to 200 µCi/µmol of purified 64 Cu-αEpCAM/αCD3 CSANs and 64 Cu-αCD3 CSANs.…”

“…However, moderate binding was seen in the monocyte population, by both the targeted and non-targeted constructs, which is validated by our previous results indicating macrophages slowly take up CSANs over time. 30…”

“…26 We have previously incorporated a variety of scFvs and small peptide sequences onto the C-termini of our DHFR 2 proteins (DHFR 2 -scFv), thereby enabling the CSANs to target specific overexpressed receptors. 27–30 Additionally, CSAN size, and therefore overall scFv valency, can be varied by modifying the length of the peptide linker between the two DHFR proteins. 31 PAR therapy has several innovations, such as the capability of quickly labeling T cell membranes in a matter of minutes and is stable on the cell surface for multiple days.…”

Harnessing self-assembled peptide nanoparticles in epitope vaccine design

“…Molecular engineering of proteins as the monomeric building blocks through self-assembly was first suggested by Drexler (1981), who foresaw the development of advanced molecular machineries and the construction of devices and materials with atomic precision based on this bright new idea. Since then, self-assembled peptides have been employed to produce a broad range of materials, such as nanofibers (Yanlian et al, 2009;Zhang, 2003), nanowires (Sun et al, 2016), nanorings (Carlson et al, 2006;Shah et al, 2016), nanotubes (Hartgerink et al, 1996;Scanlon and Aggeli, 2008), and nanoparticles (Kaba et al, 2009;Raman et al, 2006), among others. They may also form some ordered nanoscale structures at mesoscopic and macroscopic levels (Whitesides and Grzybowski, 2002), including monolayers, hydrogels, and three-dimensional scaffolds .…”

Recent advances in the bioanalytical methods of polyethylene glycols and PEGylated pharmaceuticals

“…Since the intrinsic labeling does not alter the structure of the molecule and possesses fast identical physicochemical property to analytes, it can represent the pharmacokinetic behavior of the analytes precisely. Radiolabeling is commonly used in the studies of the in vivo absorption, distribution, metabolism, and excretion (ADME) processes of PEG [62,78,79], PEGylated small molecules [80], PEGylated peptides [81–83], PEGylated proteins [84–86], and PEGylated nanoparticles [10,87–93], which will be discussed in the following parts respectively.…”