Biochemical and Biological Characterization of a New Oxidized Avidin with Enhanced Tissue Binding Properties

Abstract

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff's base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected 111 In-Biotin-DOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.

Enhanced structural stability of Concholepas hemocyanin increases its immunogenicity and maintains its non‐specific immunostimulatory effects

“…In fact, the less digestible forms of identical antigens are more immunogenic, inducing better T-and B-cell responses [17]. Some authors have reported that the introduction of reactive aldehydes groups either directly or indirectly by cross-linking reagents induces the stabilization of the structure of diverse antigens [17][18][19]. Among these agents, sodium periodate indirectly introduces aldehydes through the oxidation of the sugar moieties present in the antigen.…”

Therapeutic efficacy of intra‑tumor AvidinOX and low systemic dose biotinylated cetuximab, with and without cisplatin, in an orthotopic model of head and neck cancer

“…There is a need to develop local treatments for cancer lesions that are derived from or reside within tissues/organs that are very different in terms of stromal composition, vascularization, density and other features. AvidinOX has been previously shown to form Schiff's bases with proteins in a variety of tissues and in several animal species (7)(8)(9)(10)(11)(12)(13). Long tissue residence of AvidinOX is also currently being confirmed in human patients with liver metastases and other inoperable tumor lesions as indicated by selective and consistent uptake of intravenous 177 Lu-ST2210 administered 1 and up to 15 days after intra-tumor AvidinOX (ClinicalTrials.gov NCT02053324 and NCT03188328, data not shown).…”

“…We recently described a novel method that allows suboptimal doses of biotinylated agents to become effective therapies in models of HNC. The treatment is based on the intra-tumor injection of AvidinOX, an oxidized avidin derivative that exhibits the distinctive property of forming Schiff's bases with tissue proteins (7)(8)(9)(10)(11), followed by systemically administered radioactive biotin (12) or biotinylated cetuximab (bCet) (13). In the present study, we show that the injection of AvidinOX into FaDu pharynx squamous cell carcinoma cells xenografted in the mouse tongue increases the anti-tumor activity of a suboptimal bCet dose administered intraperitoneally after 24 h. The data also indicate that the treatment can be repeated at least twice, 1 week apart, and that the therapeutic efficacy could be further improved by including a low dose of cisplatin in the protocol.…”

Anti-ErbB2 immunotherapeutics: struggling to make better antibodies for cancer therapy

“…62 Attempts to uniquely improve trastuzumab efficacy are also coming from our group at Alfasigma SpA. From our long experience in tumor pre-targeting based on the exploitation of the avidin-biotin interaction, [63][64][65][66][67][68][69] we recently found that by anchoring biotinylated trastuzumab or pertuzumab to AvidinOX (modified avidin chemically reacting with tissue proteins)-treated tumor cells, antibody reactivity is increased by several orders of magnitude. 70 This peculiar phenomenon, which is related to the inhibition of the targeted receptor trafficking, could allow significant reduction of antibody doses with consequent reduction of toxicity and costs.…”