Janet A. Mercer-Smith

Labeling antibodies with copper radionuclides using N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine

Antibody conjugates labeled with copper-64 and -67 (64Cu and 67Cu) were prepared using the porphyrin chelator N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine (N-bzHCS3P). N-bzHCS3P was chosen because it has only one carboxylate group available for activation and coupling to antibody. The conjugates were characterized with respect to (1) the location of the porphyrin on the antibody, (2) the retention of immunoreactivity, and (3) the serum stability of the amide bond linking porphyrin to antibody. These studies showed that porphyrin attachment on the antibody surface is random. The conjugates exhibited high retention of immunoreactivity and reasonable serum stability for potential application in nuclear medicine.

Preparation and biodistribution of copper-67-labeled porphyrins and porphyrin-A6H immunoconjugates.

The synthetic porphyrins, N-benzyl-5,10,15,20-tetrakis (4-carboxyphenyl) porphine (N-bzHTCPP) and N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine (N-bzHCS3P), represent excellent radiocopper chelating agents that may find utility in antibody-mediated diagnosis and/or therapy. N-bzHCS3P was conjugated to an anti-renal cell carcinoma (RCC) antibody, A6H, and labeled with copper-67. 67CuCS3P-A6H was studied for its biodistribution in human RCC xenograft-bearing nude mice, along with the radiolabeled free porphyrins. The porphyrins resulted in tumor:blood ratios in the range of 3 to 4 after 48 h. The radiolabeled antibody achieved a tumor:blood ratio of over 16 after 45 h, indicating accumulation at the desired site. However, unwanted localization also occurred in the liver and spleen, which will have to be rectified before realizing the full potential of this approach.

A model for the origin of photosynthesis--III. The ultraviolet photochemistry of uroporphyrinogen.

Abstract— The photochemical ramifications of the high ultraviolet flux on the primordial earth prior to the formation of the ozone layer have been considered in a study of the ultraviolet photochemistry of uroporphyrinogen (urohexahydroporphyrin), a colorless compound which absorbs strongly at wavelengths less than 220 nm. Urohexahydroporphyrin was investigated since it is the first macrocycle formed on the biosynthetic pathway of chlorophyll and can be used to test the hypothesis that the biosynthetic pathway to chlorophyll recapitulates the evolutionary history of photosynthesis.

The biological characteristics of a water soluble porphyrin in rat lymph nodes

The biological characteristics of a radiolabeled metalloporphyrin, 5,10,15,20-tetrakis(4-carboxyphenyl)-porphinato [67Cu]copper (II) [( 67Cu]TCPP), in rat lymph nodes, surrounding muscle, fat, and blood were determined. Lymphatic tissue localized greater amounts of [67Cu]TCPP than did surrounding muscle and fat. Inflamed lymph nodes localized greater amounts of [67Cu]TCPP than did noninflamed lymph nodes. Time course studies suggest that the uptake of [67Cu]TCPP in noninflamed and in inflamed lymph nodes may involve different biological processes. The affinity of [67Cu]TCPP for inflamed lymph nodes may be influenced by the degree of inflammation. If further studies confirm these results, [67Cu]TCPP may be useful as a potential radiopharmaceutical for imaging inflamed lymph nodes.

The Biodistribution of Radiocopper-Labeled Compounds

Porphyrins form extremely stable chelates with Cu2+. Two copper radionuclides, 67Cu and 64Cu, have attractive nuclear decay properties for use in nuclear medicine applications. We have investigated the use of radiocopper-labeled porphyrins for localization in inflamed tissue and for attachment to antibodies for tumor imaging and therapy. We have examined the biodistribution of a 67Cu labeled porphyrin, [5, 10, 15, 20-tetrakis(4-carboxyphenyl) porphinato [67Cu] copper (II)], 67 CuTCPP. The 67CuTCPP was intravenously injected into the tail vein of Fischer F344 male rats. The kidneys, liver, and spleen localize the greatest amounts of 67CuTCPP. The elimination of 67CuTCPP from the body is described by a normal exponential decay curve with a biological half-life of 108 hours and an effective half-life of 32 hours. We have also examined the biodistribution of 5-(4-carboxyphenyl)-10, 15,20-tris(4-sulfophenyl) porphinato [67Cu] copper (II) anti-Thy 1.2 antibody conjugates in normal and tumor-bearing male AKR/J mice. The liver, kidney, and tumor have the highest uptake of the 67Cu labeled antibody conjugate. In all 67Cu labeled compounds studied, the blood clearance was rapid and the bone concentration of the radiolabeled species was low.

Effect of chemical modification strategy on the characteristics of copper-67-Labeled immunoconjugates, Part I: Immunoreactivity

AbstractThe synthetic porphyrins N-benzyl-5,10,15,20-tetrakis(4-carboxyphenyl)porphine (N-bzHTCPP) and N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl)porphine (NbzHCS3P) represent excellent radiocopper chelating agents that may find utility in antibody-mediated diagnosis and/or therapy. Detailed analyses were performed to explore the effect of the chemical modification strategy on the characteristics of immunoconjugates prepared from the anti—renal cell carcinoma monoclonal antibody A6H and N-bzHCS3P. The parameters included in the study were antibody type [intact A6H and two of its fragments, half A6H and A6H-F(ab’)2], chemical linkage type and site, the presence or absence of intermediate linker molecules, and the nature of the chemical modification steps employed. The immunoconjugate synthesized by directly coupling N-bzHCS3P to whole antibody retained 75–85% of the immunoreactivity of the unmodified antibody. In general, however, immunoconjugates prepared using the fragments or the in...

Radiometallating Antibodies and Autoantigenic Peptides

We have developed methods to radiolabel large molecules, using porphyrins as bifunctional chelating agents for radiometals. The porphyrins are substituted with an N-benzyl group to activate them for radiometallation under mild reaction conditions. Porphyrins that have one functional group for covalent attachment to other molecules cannot cause crosslinking. We have examined the labeling chemistry for antibodies and have developed methods to label smaller biologically active molecules, such as autoantigenic peptides (fragments of the acetylcholine receptor), which are pertinent to myasthenia gravis research. The methods of covalent attachment of these bifunctional chelating agents to large molecules, the radiometallation chemistry, and biological characterization of the radiolabeled compounds will be discussed.

Similarity of copper and technetium binding sites in human IgG

Kits for direct labeling of IgG with 99mTc were used without modification for the preparation of [67Cu]IgG. The IgG was pre-treated to generate thiolate groups which would bind 67Cu. The direct labeling of reduced IgG with 67Cu was highly efficient, resulting in approx. 95% 67Cu binding. Non-reduced IgG (negative control) had labeling efficiencies of less than 10%. IgG pre-exposed to Cu(II) had reduced amounts of 99mTc bound to it. The results demonstrate a direct relationship between copper- and 99mTc-binding sites in IgG.

Copper-67 labeled porphyrin localization in inflamed tissue

A series of experiments compared the uptake of 5,10,15,20 tetrakis(4-carboxyphenyl) porphinato [67Cu] copper (II), 67CuTCPP, by the lymph nodes of inflamed and two sets or control rats. The results demonstrate that 67CuTCPP localizes in greater concentration in inflamed lymph nodes than in noninflamed control lymph nodes. This enhanced uptake of 67CuTCPP by inflamed lymph nodes was 3.6 times greater than was the uptake by control lymph nodes. A time course study demonstrated that the uptake of 67CuTCPP by inflamed lymph nodes reached the maximum level by 24 hours post-injection of 67CuTCPP and remained constant throughout the 96 hours examined. It was also found that the uptake of 67CuTCPP by inflamed lymph nodes was not exclusively dependent upon an increase in the weight of inflamed lymph nodes. These studies show that 67CuTCPP has potential as a lymphoscintigraphy agent.