The study of biological macromolecules using perturbed angular correlations of gamma radiation.
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Animals↗
Biomedical subjects
C F Meares
Publications and source records attributed to C F Meares.
The study of 111 In-labeled compounds in mice, using perturbed angular correlations of gamma radiations.
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Acetates↗
Study of carbonic anhydrase using perturbed angular correlations of gamma radiation.
The angular correlation of the 150-247 kev gamma-ray cascade of (111m)Cd is strongly perturbed when this nucleus is bound to the enzyme carbonic anhydrase. A comparison of the perturbed angular correlation for the apoenzyme with that for native carbonic anhydrase confirms that the (111m)Cd binds at the active region of the enzyme. These results provide good evidence that the perturbed angular correlation reflects the effective molecular rotational correlation time at the metal binding site, and that this radioactive nucleus can be used as a rotational tracer to label biological macromolecules. The qualitative dependence of the perturbed angular correlation of the (111m)Cd cascade on the molecular rotational correlation time at the metal binding site is illustrated using a cadmium-complex solution at various temperatures.
Binding Sites↗
Simplified method for conjugating macrocyclic bifunctional chelating agents to antibodies via 2-iminothiolane.
A one-step method for conjugating macrocyclic chelators to antibodies using the protein modification reagent 2-iminothiolane controls aggregation, maintains immunoreactivity, and produces consistent chelate/antibody ratios. Conjugation conditions have been investigated with the macrocyclic chelates 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N ',N",N"'-tetraacetic acid and 2-[p-(bromoacetamido)benzyl]-1,4,7,10-tetraazacyclododecane-N,N',N ",N"'-tetraacetic acid, with three different monoclonal antibodies. The bifunctional chelating agents are prepared by bromoacetylation of their amine precursors using a two-phase H2O/CHCl3 system, which improves product purity.
Antibodies, Monoclonal↗
N-terminal modification of immunoglobulin polypeptide chains tagged with isothiocyanato chelates.
Conjugates of monoclonal antibodies with drugs, toxins, radionuclides, and other agents are in widespread use in therapeutic trials and as clinical research tools. The characterization of these immunoconjugates generally does not include determining the individual sites at which such agents are attached. We have begun to explore the attachment of the bifunctional chelating agent isothiocyanatobenzyl-EDTA (CITC1) to the N-termini of the light chains of the Lym-1 monoclonal antibody. The similarity between this bifunctional chelating agent and Edman's reagent, phenyl isothiocyanate, led us to develop methods to distinguish between chelate-conjugated alpha-amino groups and epsilon-amino groups by Edman degradation. Practically all the N-terminal Asp alpha-NH2 groups of Lym-1 can be modified at neutral pH, while attachment at lysine side chains predominates at pH 9. Comparison of the immunoreactivities of Lym-1-CITC conjugates with and without N-terminal conjugation shows that both are almost fully active. This implies that modification of light-chain N-termini has little or no effect on immunoreactivity, despite the fact that these residues lie near the antigen-binding sites.
Amino Acid Sequence↗
Synthesis of novel 1,4,7-triazacyclononane-N,N',N"-triacetic acid derivatives suitable for protein labeling.
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Amino Acid Sequence↗
A convenient and flexible approach for introducing linkers on bifunctional chelating agents.
The synthesis of a protected bifunctional analog of ethylenediaminetetraacetic acid (EDTA) is described. The molecule contains an aminobenzyl moiety that allows the easy attachment of the chelating agent to a wide variety of groups. Examples of reaction with the C-termini of two peptides are given. In the following paper, the two peptides are used to study the enzymatic cleavage of metal chelates from a monoclonal antibody.
Amino Acid Sequence↗
Influence of a peptide linker on biodistribution and metabolism of antibody-conjugated benzyl-EDTA. Comparison of enzymatic digestion in vitro and in vivo.
Insight into the metabolism of radiolabeled antibodies is important for the design of better radioimaging and therapy agents. To test the effect of linkers that can be cleaved in vivo, we introduced Ala-Leu-Ala-Leu between the antibody Lym-1 and an 111In-labeled benzyl-EDTA. For comparison, we studied a conjugate without the linker. Digestion of the two conjugates in vitro showed that the one with Ala-Leu-Ala-Leu was cleaved rapidly by the liver protease cathepsin B1 (T1/2 approximately 6 h). After 100 h of digestion, reversed-phase HPLC product analysis of the Ala-Leu-Ala-Leu conjugate showed that 48% of the total radioactivity had the same retention time as (p-aminobenzyl)-EDTA(In), and 37% of the total radioactivity had the same retention time as [p-(Ala-Leu-amido)benzyl]-EDTA(In). After 97 h of digestion, the conjugate without the linker had 79% of the radioactivity activity still attached to the protein. We also tested the two conjugates in mice. Ala-Leu-Ala-Leu had only a moderate effect on the whole body and liver clearance in vivo. The excretion of the radioactivity was about 6% per day with the linker and about 3% per day without the linker. HPLC analysis of the urine of a single mouse showed products similar to the in vitro study; 54% of the excreted radioactivity had the same retention time as (p-aminobenzyl)-EDTA(In), while 10% had the retention time of [p-(Ala-Leu-amido)benzyl]-EDTA(In).
Amino Acid Sequence↗
Large-scale synthesis of the bifunctional chelating agent 2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetr aacetic acid, and the determination of its enantiomeric purity by chiral chromatography.
The attachment of radiometals to monoclonal antibodies for medical applications requires extreme stability under physiological conditions, with no significant release of metal. Chelators that can hold radiometals like 111In, 67Ga, and 90Y with high stability under these conditions are essential for radiotherapy or immunoscintigraphy. 2-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclododecane- N,N',N'',N'''-tetraacetic acid (nitrobenzyl-DOTA) is one of the most promising bifunctional chelating agents. A large-scale synthesis of nitrobenzyl-DOTA is described. The overall yield for the nine-step synthesis sequence starting from nitrophenylalanine is 5.6%. Synthesis of nitrobenzyl-DOTA according to the new procedure yields up to approximately 10 g without special apparatus. Both enantiomers of the chiral chelate nitrobenzyl-DOTA have been prepared, and their enantiomeric purity has been checked by chiral chromatography.
Chelating Agents↗
Synthesis, metal chelate stability studies, and enzyme digestion of a peptide-linked DOTA derivative and its corresponding radiolabeled immunoconjugates.
By directly coupling a tetrapeptide to DOTA through an amide bond, we synthesized a novel DOTA derivative, DOTA-glycylglycylglycyl-L-p-nitrophenylalanine amide. We converted this new precursor bifunctional chelating agent to DOTA-glycylglycylglycyl-L-p-isothiocyanatophenylalanine++ + and conjugated it to monoclonal antibody Lym-1. Serum stability studies show that the radiolabeled conjugates are kinetically inert under physiological conditions. The rates of loss of radiometals in human serum are 0.1 +/- 0.1% per day for InIII, 0.02 +/- 0.15% per day for YIII, and 0.3 +/- 0.2% per day for CuII labeled immunoconjugates. In the presence of the liver enzyme cathepsin B, an in vitro digestion of 114mIn-labeled conjugate yields a small fragment containing 114mIn. Characterization of the cleavage products shows that this liver enzyme hydrolyzes the peptide linkage before the phenylalanine residue, freeing the In-DOTA-triglycine complex from the conjugate. However, the liver enzyme cathepsin D does not cleave the linkage over the span of 7 days.
Amino Acid Sequence↗
Labeling monoclonal antibodies with 90yttrium- and 111indium-DOTA chelates: a simple and efficient method.
Yttrium-90 and indium-111 have been attached to a monoclonal antibody with a bifunctional chelating agent (DOTA-peptide). Using the unique features of this DOTA-peptide and its complexes with trivalent yttrium and indium, the bifunctional chelating agent was prelabeled with either radiometal and then conjugated to chimeric monoclonal antibody L6. Both radiolabeling procedures and yield are suitable for the practical preparation of radiopharmaceuticals. Biodistribution studies in tumor-bearing mice showed that, e.g., on day 3 after intravenous injection of a 90Y immunoconjugate, liver uptake was 5.4 +/- 1.5% ID/g, bone uptake 2.0 +/- 0.5% ID/g, and tumor uptake 18.0 +/- 8.0% ID/g.
Animals↗
Improved synthesis of 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane- N,N',N",N"-tetraacetic acid and development of a thin-layer assay for thiol-reactive bifunctional chelating agents.
Monoclonal antibodies labeled with radiometals such as copper-67 have applications in radioimmunodiagnosis and radioimmunotherapy. Moi et al. [(1985) Anal. Biochem. 148, 249-253] showed that 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane- N,N',N",N"-tetraacetic acid (BAT) is an effective reagent for linking copper to proteins, and antibodies labeled with copper radionuclides are currently undergoing clinical trials. Here we describe improvements in the original synthesis that increase the overall yield of BAT to 23%. We also describe a new assay useful to determine the activity of bifunctional chelating agents with thiol-reactive functional groups.
Chelating Agents↗
Synthesis of the protein cutting reagent iron (S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate and conjugation to cysteine side chains.
Convenient methodology for preparation and conjugation of the protein-cutting iron chelate iron (S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) is given. This formulation of the reagent can be handled in a manner analogous to many other protein-labeling reagents, such as fluorescent probes or cross-linkers. By taking advantage of the recently discovered peptide hydrolysis reaction, the chelate may be tethered to a single site (e.g., a cysteine side chain) and used to map its proximity to individual peptide bonds by automated Edman sequencing of the protein fragments produced. The method is illustrated by conjugation of Fe-BABE to the carboxy terminal domain (amino acid residues 234-329) of the Escherichia coli RNA polymerase alpha subunit. The molecular mass of the protein conjugate was confirmed by electrospray ionization mass spectrometry.
Chromatography, High Pressure Liquid↗
Cathepsin substrates as cleavable peptide linkers in bioconjugates, selected from a fluorescence quench combinatorial library.
Several extended peptide substrates for the human liver enzymes cathepsin B and cathepsin D have been selected as cleavable linkers for lysosomal proteolysis of bioconjugates. A one-bead-one-peptide combinatorial library of 9(4) fluorogenic substrates was employed. We designed this library to explore a set of substrates containing nonionizable/nonoxidizable groups to meet the requirements of prelabeling [Li et al. (1994) Bioconjugate Chem. 5, 101-104] as well as to yield stable conjugates whose preparation is straightforward.
Acrylamides↗
Total solid-phase synthesis of 1,4,7,10-tetraazacyclododecane-N,N', N'',N'''-tetraacetic acid-functionalized peptides for radioimmunotherapy.
A convenient approach to the functionalization of peptides with the macrocyclic 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) moiety has been developed. Protected components (using tert-butyl or tert-butyloxycarbonyl groups) of both the peptide and the chelate were assembled on the same solid resin support. Deprotection and cleavage of the resin-bound DOTA-peptides were performed in one step using a trifluoroacetic acid cleavage mixture to yield free DOTA-peptide amides.
Amino Acid Sequence↗
Enzymatic cleavage of peptide-linked radiolabels from immunoconjugates.
We have incorporated peptides selected by combinatorial library [Peterson, J. J., and Meares, C. F. (1998) Bioconjugate Chem. 9, 618-626) into peptide-linked radiolabeled immunoconjugates of the form DOTA-peptide-antibody. Decapeptide linkers -GFQGVQFAGF- and -GFGSVQFAGF-, selected for cleavage by human liver cathepsin B, were rapidly digested in vitro when compared to the simple model tetrapeptide motif of the prototype -GGGF- [Li, M., and Meares, C. F. (1993) Bioconjugate Chem. 4, 275-283]. Cleavage properties of these library-selected substrates for cathepsin B compared favorably with decapeptide linkers -GLVGGAGAGF- and -GGFLGLGAGF-, which incorporate two of the most labile extended cathepsin B substrates from the literature. The decapeptide linker -GFGSTFFAGF-, selected from the library for cleavage by human liver cathepsin D, was rapidly digested by cathepsin D while the others were not.
Cathepsin B↗
Efficient multigram synthesis of the bifunctional chelating agent (S)-1-p-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid [correction of diethylenetetraminepentaacetic acid].
We have developed and optimized the synthesis of the title compound, eliminating all HPLC purifications prior to the final product. The yield (and scale) of the synthesis was increased from 19% (200 mg) to 75% (26 g).
Antibodies, Monoclonal↗
Antibodies against metal chelates.
Because monoclonal antibodies can recognize and bind to specific groups of atoms such as tumour antigens, they have promise for use in vivo as carriers of radionuclides, drugs or other appended molecules for diagnosis and treatment of disease. Attachment of metal ions to antibodies by means of bifunctional chelating agents can add the diverse nuclear, physical and chemical properties of the metallic elements to these specific binding proteins (ref. 4 and refs therein). With the ultimate aim of engineering probe-binding properties into the antibodies themselves, we have now prepared monoclonal antibodies against the EDTA chelate of indium. These antibodies show a remarkable preference for indium chelates; changing to another metal such as scandium or gallium can decrease the antibody-binding constant by more than three orders of magnitude. These antibodies also introduce a new degree of control over the biological distributions of chelated radionuclides, markedly altering their uptake in tumours and normal organs.
Antibodies, Monoclonal↗