Site-Specific Conjugation of Monodispersed DOTA-PEGn to a Thiolated Diabody Reveals the Effect of Increasing PEG Size on Kidney Clearance and Tumor Uptake with Improved 64-Copper PET Imaging
| Autor: | Peter J. Hudson, Fabio Turatti, John E. Shively, Erasmus Poku, Lin Li, Anne-Line Anderson, David Tai Leong, Michael Paul Wheatcroft, Desiree Crow, David Colcher, James R. Bading, Jenny Carmichael, Andrew Raubitschek, Paul J. Yazaki |
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| Rok vydání: | 2011 |
| Předmět: |
Biomedical Engineering
Mice Nude Pharmaceutical Science chemistry.chemical_element Bioengineering Conjugated system Kidney Article Polyethylene Glycols Heterocyclic Compounds 1-Ring Mice chemistry.chemical_compound Neoplasms PEG ratio medicine Animals DOTA Tissue Distribution Sulfhydryl Compounds Binding site Pharmacology Binding Sites Molecular Structure biology Chemistry Organic Chemistry Copper medicine.anatomical_structure Copper Radioisotopes Biochemistry Positron-Emission Tomography biology.protein Biophysics Female Radiopharmaceuticals Antibody Neoplasm Transplantation Biotechnology Conjugate |
| Zdroj: | Bioconjugate Chemistry. 22:709-716 |
| ISSN: | 1520-4812 1043-1802 |
| DOI: | 10.1021/bc100464e |
| Popis: | Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t(1/2) = 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope (64)Cu (t(1/2) = 12 h). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site-specific attachment of monodispersed PEGn of increasing molecular size (n = 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake, and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were preconjugated to the chelator DOTA, the conjugates were able to bind radiometals such as (111)In and (64)Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80%ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24 h when radiolabeled with (111)In and 37.9% ID/g of tumor uptake (T/B = 8) at 44 h when radiolabeled with (64)Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 h observed with diabodies that were pegylated on surface lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cys-conjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting. |
| Databáze: | OpenAIRE |
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