Conjugation of metallothionein to a murine monoclonal antibody

Brown, Beverly A.; Drozynski, Cynthia A.; Dearborn, Cynthia B.; Hadjian, Richard A.; Liberatore, Frederick A.; Tulip, T. H.; Tolman, Glen L.; Haber, Stephen B.

doi:10.1016/0003-2697(88)90406-x

Cited by 17 publications

(5 citation statements)

References 9 publications

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“…When used with MAb fragments, N 3 S-type or, alternatively, the hydrazinopyridine chelating agents have required the incorporation of special cleavable linkers between chelate and protein in order to reduce nontarget accumulation of radioactivity (22,23). Derivatization of antibodies with metallothionein (a 99m Tc-chelating entity) has been reported to lead to aggregation, requiring careful preparative purification (24). Direct labeling methods, applicable to the 99m Tc labeling of an IgG or a Fab′ fragment, are problematic with F(ab′) 2 fragments in that substantial formation of 99m Tc-Fab′ occurs under labeling conditions (25)(26)(27).…”

Section: Discussionmentioning

confidence: 99%

Thiolations, ^99mTc Labelings, and Animal In Vivo Biodistributions of Divalent Monoclonal Antibody Fragments

et al. 1996

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A number of divalent monoclonal antibody fragments have been derivatized with a bifunctional reagent containing a latent thiol group, and the conjugates were thiol-deprotected by exposure to aqueous hydroxylamine. This two-step procedure enabled convenient 99mTc labeling of thiolated MAb divalent fragments, using either performed 99mTc-glucoheptonate or [99mTc] pertechnetate and single-vial lyophilized formulations of the conjugate and tin. Nonspecific incorporation of radiolabel was negligible, if any, when using nonthiolated antibodies. In animal biodistributions, in nude mice bearing LS174T human colon carcinoma xenografts, the 99mTc-labeled MAb divalent fragments studied were found to result in a 3-fold reduction in the kidney uptake of radioactivity at 24 h, compared to a 99mTc-Fab, and also to lead to improvements in other tumor:nontumor ratios.

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Section: Discussionmentioning

confidence: 99%

Thiolations, ^99mTc Labelings, and Animal In Vivo Biodistributions of Divalent Monoclonal Antibody Fragments

et al. 1996

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“…Although glucoheptonate was used successfully to transchelate reduced 99mTc to a metallothionein-conjugated antibody (11), in this investigation, citrate was first considered as a transchelator because it has been used successfully in other thiol-based labeling procedures using radiorhenium (21,22). Whereas it was possible to radiolabel with 188Re unconjugated horse-kidney metallothionein in this manner, citrate failed to provide a label on metallothionein-streptavidin.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, metallothionein, free from cadmium and saturated with zinc to protect the free sulfhydryls, was conjugated to the B72. 3 IgG antibody using a heterobifunctional crosslinking agent and effectively radiolabeled with 99mTc (11).…”

Section: Introductionmentioning

confidence: 99%

Recombinant Metallothionein-Conjugated Streptavidin Labeled with 188Re and 99mTc

Virzi¹,

Winnard²,

Fogarasi³

et al. 1995

Bioconjugate Chem.

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Consideration is now being given to the use of avidin (or streptavidin) and biotin for radiotherapy of tumor. Accordingly, the goal of this study was to radiolabel a mouse metallothionein-streptavidin fusion protein with 188Re and to compare its properties to those of the same fusion protein radiolabeled with 99mTc. A recombinant metallothionein-streptavidin fusion protein was radiolabeled by transchelation with 99mTc- and 188Re-glucoheptonate. Labeling efficiency, which was not optimized for either radionuclide, was approximately 60% for 99mTc and 20% for 188Re. Radiochemical purity was demonstrated by size exclusion HPLC both by nearly quantitative shifts of the 188Re label to higher molecular weight upon the addition of biotinylated antibody and by the absence of a shift with biotinsaturated 188Re-metallothionein-streptavidin. Stability of the labels in 37 degrees C serum was evaluated by comparing the HPLC radiochromatograms of serum samples both before and after the addition of biotinylated antibody. The 188Re label behaved like 99mTc in that the same peaks were evident, including one prominent peak due to labeled cysteine. Recoveries during HPLC analysis of serum samples showed that oxidation rates to perrhenate and pertechnetate were identical. However, instability to cysteine challenge was greater for 188Re; for example, the loss of label to cysteine after 24 h under one set of conditions was 41% for 188Re and 22% with 99mTc. Analysis by HPLC of liver and kidney homogenates from mice administered the labeled antibodies were qualitatively and, in large measure, quantitatively independent of label. Biodistributions at 5 h in normal mice were statistically identical between the two labels in blood and in most tissues. In conclusion, streptavidin may be radiolabeled with radiorhenium using recombinant mouse metallothionein as a bifunctional chelator, and under one set of labeling conditions at least, 188Re showed similar in vitro and in vivo behavior to that of 99mTc labeled to the same fusion protein.

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“…Studies of MTs with technetium are scarce; most of them were performed around the 90s and are usually based on the metal-binding capability of MTs and the possible conjugation of the protein to a biomolecule. It allows that MTs can act as bifunctional chelating agents linking the radionuclide to biomolecules [15][16][17][18][19]. As far as we know, only two of those works are essentially devoted to the study of the binding ability of MTs to technetium [20,21].…”

Section: Introductionmentioning

confidence: 99%

Rhenium and technetium tricarbonyl, {M(CO)3}+ (M = Tc, Re), binding to mammalian metallothioneins: new insights into chemical and radiopharmaceutical implications

et al. 2014

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This paper deals with the binding of the four mammalian metallothioneins (MTs) to the organometallic metal fragment {fac-M(CO)3}(+) (M = (99)Tc, Re), which is highly promising for the preparation of second-generation radiopharmaceuticals. The study of the transmetallation reaction between zinc and rhenium in Zn7-MT1 by means of UV-vis and CD spectroscopy demonstrated the incorporation of the {fac-Re(CO)3}(+) fragment to the MTs. This reaction should be performed at 70 °C to accelerate the reaction rate, a result that is consistent with the reported reactivity of the rhenium fragment. ESI-TOF MS demonstrated the formation of mixed-metal species as Zn6,{Re(CO)3}-MT, Zn6,{Re(CO)3}2-MT, and Zn5,{Re(CO)3}3-MT, as well as the different reactivity of the four MT isoforms. Hence, Zn-MT3 showed the highest reactivity, in agreement with its high Cu-thionein character, whereas Zn-MT2 exhibited the lowest reactivity, in line with its high Zn-thionein character. The reactivity of the Zn-loaded forms of MT1 and MT4 is intermediate between those of MT3 and MT2. The study of the binding of the {fac-(99)Tc(CO)3}(+) fragment to MTs showed a significant and very interesting different reactivity in relation to rhenium. The transmetallation reaction is much more effective with technetium than with rhenium and significant amounts of mixed Zn x ,{(99)Tc(CO)3} y -MT species were formed with the four MT isoforms whereas only MT3 rendered similar amounts of rhenium derivatives. The results obtained in this study support the possible use of technetium for labelling mammalian metallothioneins and also for possible radiopharmaceutical applications.

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Conjugation of metallothionein to a murine monoclonal antibody

Cited by 17 publications

References 9 publications

Thiolations, ^99mTc Labelings, and Animal In Vivo Biodistributions of Divalent Monoclonal Antibody Fragments

Thiolations, ^99mTc Labelings, and Animal In Vivo Biodistributions of Divalent Monoclonal Antibody Fragments

Recombinant Metallothionein-Conjugated Streptavidin Labeled with 188Re and 99mTc

Rhenium and technetium tricarbonyl, {M(CO)3}+ (M = Tc, Re), binding to mammalian metallothioneins: new insights into chemical and radiopharmaceutical implications

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Conjugation of metallothionein to a murine monoclonal antibody

Cited by 17 publications

References 9 publications

Thiolations, 99mTc Labelings, and Animal In Vivo Biodistributions of Divalent Monoclonal Antibody Fragments

Thiolations, 99mTc Labelings, and Animal In Vivo Biodistributions of Divalent Monoclonal Antibody Fragments

Recombinant Metallothionein-Conjugated Streptavidin Labeled with 188Re and 99mTc

Rhenium and technetium tricarbonyl, {M(CO)3}+ (M = Tc, Re), binding to mammalian metallothioneins: new insights into chemical and radiopharmaceutical implications

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Thiolations, ^99mTc Labelings, and Animal In Vivo Biodistributions of Divalent Monoclonal Antibody Fragments

Thiolations, ^99mTc Labelings, and Animal In Vivo Biodistributions of Divalent Monoclonal Antibody Fragments