Specific and stable labeling of antibodies with technetium-99m with a diamide dithiolate chelating agent.

Abstract

Technetium-99m labeling of antibodies has been suboptimal because of low affinity adventitious binding, nonspecific labeling, and loss of immunoreactivity. The diamide dithiolate ligand system (N2S2) forms highly stable, well-defined tetradentate complexes with Tc(V). Antibodies and their fragments have been labeled by conjugation of preformed "mTc4,5-bis(thioacetamido)pentanoate active ester to protein amine groups to give a chemically known 99"Tc-N2S2 complex covalently linked to antibody. Evaluations of the "'Tc-N2S2-bound antibodies and their fragments have shown high stability and retained immunoreactivity.Successful targeting ofdiagnostic radionuclides to tumors not only provides a tool to diagnose and stage cancer but also demonstrates feasibility for therapy where ligand systems can be applied to therapy radionuclides. Early studies with radiolabeled antibodies utilized radioiodine (1231/1311) because of extensive experience in protein radioiodination, covalent attachment, and ready availability of the radionuclide (1,2). Improved tumor-to-nontumor ratios were achieved with 1"'In compared to 131I by using diethylenetriaminepentaacetate (DTPA) bifunctional chelating agent technology (3)(4)(5) MATERIALS AND METHODS Preparation of 99mTc-4,5-bis(thioacetamido)pentanoyl (N2S2)-Conjugated Anti-Melanoma 9.2.27 F(ab')2 Fragment. To a mixture of 25 1.l of 4,5-bis(benzoylthioacetamido)pentanoic acid (1.0 mg/ml solution in 90% CH3CN) and 100 ,ul of 1 M NaOH was added 100 mCi of sodium [99mTc]pertechnetate in 1.0 ml of saline (0.9% NaCl). Then 1.0 mg of sodium dithionite (0.10 ml of a freshly prepared 10 mg/ml solution) was added, and the mixture was heated at 750C for 15 min.The pH was brought to about 6 with 0.10 ml of 1 M HCI and 0.30 ml of 0.2 M sodium phosphate buffer (pH 6.0). Then 10.0 mg of 2,3,5,6-tetrafluorophenol (0.10 ml of a 100 mg/ml solution in 90% CH3CN) and 12.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.10 ml of a 125.0 mg/ml solution in 90% CH3CN) were added, and the solution was heated at 75°C for 30 min. The resulting tetrafluorophenyl active ester derivative of "mTc-4,5-bis(thioacetamido)pentanoate was purified by loading the reaction mixture on a conditioned C18 cartridge (J. T. Baker), washing with 2.0 ml of 20% (vol/vol) ethyl alcohol/0.01 M sodium phosphate, pH 7.0, eight times, and eluting with 100% CH3CN. The solvent was evaporated under a stream of N2. Then 0.5 ml of the 9.2.27 F(ab')2 fragment (16) at 2.5 mg/ml and 0.50 ml of 0.2 M sodium phosphate (pH 9.0) were added for conjugation. After 15 min at room temperature, 25 mg of lysine (0.25 ml of a 250-mg/ml solution at pH 9.0) was added to quench unreacted ester. The 99mTc-N2S2-9.2.27 F(ab')2 was purified by passage through a G-25 Sephadex column (Pharmacia) equilibrated with phosphate-buffered saline.Abbreviations: N2S2, diamide dithiolate chelating system; 99M Tc-N2S2-9.2.27 F(ab')2, 99mTc-4,5-bis(thioacetamido)pentanoyl-9.2.27 F(ab')2 fragments; DPTA, diethylenetriaminepentaacetate.

Design and characterization of α-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination

“…The metal-binding site must be engineered so that metal coordination does not disrupt the biologically active conformation of the molecule. The cyclic ␣-MSH analog (Cys 4,10 , D-Phe 7 )-␣-MSH 4-13 (APOMSH) was used as the starting point of our studies (24). APOMSH retains a core sequence of residues, His-D-Phe-Arg-Trp that has been found to be sufficient for the bioactivity of ␣-MSH (17).…”

“…These molecules specifically target tumor cells by virtue of their high specificities for receptors and antigens present on the surfaces of these cells. In one commonly used approach, metallic radionuclides such as 186 Re, 188 Re, and 99m Tc are appended to the tumor-targeting molecule through bifunctional chelate groups that consist of a metal chelate and an activatable crosslinker (9)(10)(11). The resulting radiolabeled proteins, peptides, and small molecules are decorated with one or more chelating groups.…”

Serum half-life and tumor localization of a chimeric antibody deleted of the CH2 domain and directed against the disialoganglioside GD2.

“…The improvement of tumor uptake ratios achieved by the CH2-deleted antibody is similar to that described by others, when comparing F(ab')2 fragments with whole murine mAbs (5,6,20,21). Rapid tumor localization and good localization ratios are properties that make CH2-deleted antibodies good candidates for radioimaging of tumors, possibly allowing the use of radioisotopes with low energy and a short half-life, such as 1231I or 991Tc (24,25). The use of CH2-deleted antibodies to deliver cytotoxic agents such as toxins, drugs, or radionuclides for therapy may be limited by its fast elimination; however, a possible exception may be the targeting of a-particle-emitting radionuclides such as 212Bi or 211At, with half-lives of 61 min and 7.2 hr, respectively (26,27).…”

Effective coupling of Re/Tc-MAG3 complexes with amines and nucleobases in aprotic solvents

“…In the pre-labeling method BFCA is coordinated with 99mTc first and the resulting complex is linked to the biomolecule in a second step. 4 This strategy assures formation of a well characterised Tc-complex avoiding nonspecific binding of Tc to the peptide derivative.…”