The purpose of this research was to optimize chemical parameters to radiolabel antibody fragments and other biologics with Tc-99m for scintigraphic detection of tumors. Tc-99m is the optimal radionuclide used for scintigraphic imaging but Tc99m labeled biologics have not been actively used mainly because of complexity of Tc-99m chemistry. We have taken a bifunctional chelate approach which involved the synthesis of a bifunctional chelator (BFC), conjugation of the BFC to biologics and radiolabeling of the chelator moiety with Tc-99m. We have selected mercaptoacetyltriglycine (MAG3) as the BFC agent because it binds Tc-99m strongly forming a square pyramid structure used as a renal imaging agent. To use MAG3 as a BFC, we had to overcome two major obstacles. One was related to the chemistry of mercapto group. The mercapto group is known to be the best ligand for binding Tc-99m. However, it is also one of the most chemically reactive groups. As a result, we had to find a right protecting group which protects the mercapto group from undergoing chemical reactions during storage but is readily de- protected under a mild condition. The second problem was related to Tc-99m chemistry. Technetium-99m available from a Tc-99m generator is in the form of Tc-99m pertechnetate which is chemically non-reactive and has to be reduced to an oxidation state of +5 for binding with MAG3. This reduced Tc-99m is very reactive and can undergo various reactions such as reoxidation to Tc-99m pertechnetate, binding with proteins non-specifically, and hydrolysis reaction to Tc-99m colloids if no chelating agent is present. Therefore, a transchelating agent had to be selected to hold both the reduced Tc-99m and stannous ion (reducing agent) in a soluble form but transfer Tc-99m to MAG3 when the mercapto group of MAG3 is generated. Using a benzoyl group as a mercapto protecting group and tricine as a transchelating agent, we have successfully labeled Bz-MAG3 conjugated antibody Fab and a single chain Fv from Dr. Ira Pastan with Tc-99m with a 50% labeling yield. The immunoreactivity and specificity of the labeled fragments will be determined in the near future.
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