Characterization of therapeutic protein AvidinOX by an integrated analytical approach

Abstract

AvidinOX, the oxidized derivative of Avidin, is a chemically modified glycoprotein, being currently under clinical investigation for targeted delivery of radioactive biotin to inoperable tumors. AvidinOX is produced by 4-hydroxyazobenzene-2-carboxylic acid (HABA)-assisted sodium periodate oxidation of Avidin. The peculiar property of the periodate-generated glycol-split carbohydrate moieties to form Schiff's bases with amino groups of the tissue proteins allows to achieve a tissue half-life of 2 weeks compared to 2 h of native Avidin. Carbohydrate oxidation, along with possible minor amino acid modifications, introduces additional microheterogeneity in the glycoprotein structure, making its characterization even more demanding than for native glycoproteins. Aiming at the elucidation of the effects of oxidation conditions on the AvidinOX protein backbone and sugars, this microheterogeneous glycoprotein derivative was characterized for the first time using a combination of different analytical methods, including colorimetric methods, mass spectrometry, hollow-fiber flow field-flow fractionation with UV and multi-angle laser scattering detection (HF5-UV-MALS), and NMR. The proposed integrated approach reveals structural features of AvidinOX relevant for its biological activity, e.g., oxidized sites within both carbohydrate moieties and protein backbone and conformational stability, and will be considered as an analytical tool for AvidinOX industrial preparations. It is worth noting that this study enriches also the structural data of native Avidin published up-to-date (e.g., glycan structure and distribution, peptide fingerprint, etc.). Graphical abstract Scheme of phenylacetic hydrazide/MALDI-TOF approach for quantification of aldehydes in AvidinOX based on the determination of the number of hydrazone adducts between hydrazide reagent and aldehyde groups of protein.

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.…”