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Optimizing and determining the click chemistry mediated Cu-64 radiolabeling and physiochemical characteristics of trastuzumab conjugates. | LitMetric

Optimizing and determining the click chemistry mediated Cu-64 radiolabeling and physiochemical characteristics of trastuzumab conjugates.

Biochem Biophys Res Commun

Department of Molecular Medicine, Brain Korea 21, Four KNU Convergence Educational Program of Biomedical Sciences for Creative Future Talents, School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea. Electronic address:

Published: January 2023

AI Article Synopsis

Article Abstract

Over the last decade, Cu-labeling of monoclonal antibody (mAb) via inverse electron demand Diels-Alder click chemistry (IEDDA) have received much attention. Despite the tetrazine-transcyclooctene (Tz-TCO) click chemistry's convenience and efficiency in mAb labeling, there is limited information about the ideal parameters in the development of click chemistry mediated (radio)immunoconjugates. This encourages us to conduct a systematic optimization while concurrently determining the physiochemical characteristics of the model mAb, trastuzumab, and TCO conjugates. To accomplish this, we investigated a few critical parameters, first, we determined the degree of conjugations with varying molar equivalents (eq.) of TCO (3, 5, 10, and 15 eq.). Through analytical techniques like size exclusion chromatography, dynamic light scattering, and zeta potential, qualitative analysis were performed to determine the purity, degree of aggregation and net charge of the conjugates. We found that as the degree of conjugation increased the purity of intact mAb fraction is compromised and net charge of conjugates became less positive. Next, all trastuzumab-PEG-TCO conjugates with varying molar ratio and quantity (30, 50, 100, 200, 250 μg) were radiolabeled with Cu-NOTA-PEG-Tz via IEDDA click chemistry and radiochemical yields were determined by radio-thin layer chromatography. The radiochemical yields of trastuzumab conjugates improved with increased amount and molar ratio. Next, we investigated the effect of the radioprotectant ascorbic acid (AA) of varied concentrations (0.25, 0.5, 0.75, 1 mM) on radiochemical yields and subsequent pharmacokinetics. A concentration of 0.25 mM of AA was found to be optimal for click reaction and in vivo biodistribution. Finally, we investigated the indirect influence of bioconjugation buffers on radiochemical yields and biodistribution in NIH3T6.7 tumor models that resulted approximately ∼11 %ID/g tumor uptake.

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http://dx.doi.org/10.1016/j.bbrc.2022.11.026DOI Listing

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