Radioactive Labeling of Antibody: A Simple and Efficient Method

Hnatowich, Donald J.; Layne, W.W.; Childs, R.L.; Lanteigne, D.; Ma, Davis; Tw, Griffin; Doherty, P.W.

doi:10.1126/science.6836304

Cited by 374 publications

(146 citation statements)

References 7 publications

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“…The strong chelator, DTPA [13,31], is used to complex a variety of heavy metal radioisotopes, such as indium and technetium, to proteins, including monoclonal antibodies, for diagnostic and therapeutic purposes. We attempted to use the DTPA prosthetic group to make available heavy metal analogs of adenosine receptor agonists, 12, and antagonists 13.…”

Section: Discussionmentioning

confidence: 99%

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Molecular probes for extracellular adenosine receptors

Jacobson

Ukena

Padgett

et al. 1987

Biochemical Pharmacology

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Derivatives of adenosine receptor agonists (N 6 -phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341]. The "functionalized congener" approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (K i ~ 10 −9 −10 −8 M) in A 1 -receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [ 3 H]ADAC has been synthesized and found to bind highly specifically to A 1 -adenosine receptors of rat and calf cerebral cortical membranes with K D values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19 F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed."Functionalized congener" refers to a drug derivative that contains a chemical functional group, such as an amine or carboxylic acid, attached through a spacer chain to a position on the pharmacophore, which position is relatively insensitive to steric bulk. The congeners are designed as intermediates for the synthesis of covalent conjugates that retain binding properties at the receptor site for the drug. The aims of this approach are 2-fold: (1) to develop potential new pharmaceutical agents in which the activity of the primary pharmacophore (portion of drug molecule essential for biological activity) may be modulated through distal changes, and (2) to synthesize radioactive and non-radioactive molecular probes for the drug binding site. Since the strategy of this drug design allows groups to be placed at a distance from the binding site of the primary pharmacophore, it is possible to overcome constraints on the size of the attached group [1][2][3][4][5].Sites for functionalization of adenosine receptor ligands have been identified for both agonists (derivatives of N 6 -phenyladenosine) and antagonists (derivatives of 1,3-dialkyl-8-phenylxanthine . A common structure-activity relationship (SAR) feature in both agonist and antagonist series is that the presence of a distal amino group located on the attached chain in many cases results in increased affinity to adenosine receptors.In this study we have examined the parameters of binding of tritiated ADAC and have demonstrated the utility of both ADAC and XAC as synthetic precursors for receptor probes. Biotin-containing probes for adenosine receptors have been reported [6]. ...

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Section: Discussionmentioning

confidence: 99%

“…Metal complexing probes, 12 and 13, were prepared similarly using diethylenetriaminepentaacetic (DTPA) anhydride (Sigma), introduced by Hnatowich et al [13]. The Californium mass spectrum for compound 13 showed major peaks at 827 (M + Na) + and at 805 (M + H) + m.u.…”

Section: Synthesis Of Receptor Probesmentioning

confidence: 99%

Molecular probes for extracellular adenosine receptors

Jacobson

Ukena

Padgett

et al. 1987

Biochemical Pharmacology

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“…Labeling with "1'In (6) was performed by the diethylenetriaminepentaacetic acid (DTPA) cyclic anhydride method (7). The ratio of anhydride to 7E3 was 50:1 and removal of unconjugated DTPA was accomplished by exhaustive dialysis against 0.01 M Hepes/0.15 M NaCl buffer, pH 7.…”

Section: Methodsmentioning

confidence: 99%

Thrombus radioimmunoscintigraphy: an approach using monoclonal antiplatelet antibody.

Oster¹,

Srivastava²,

Som³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Thrombus detection and localization is of cardinal importance in clinical medicine. The currently available method using autologous "1In-labeled platelets is too lengthy and complex for everyday use. It requires careful separation of the platelets prior to labeling and visualization of the thrombus becomes possible only 24 hr after injection. An approach to thrombus imaging using a monoclonal antiplatelet antibody labeled with "'In or 123I is described. The antibody (7E3) prepared against human platelets inhibits the interaction between fibrinogen-coated beads and both human and dog platelets. 7E3 is an IgG1 that binds to the complexed glycoprotein Ilb/Illa. Ninety percent of a tracer dose of radiolabeled 7E3 binds to human platelets and 50% binds to dog platelets. In vitro studies showed that virtually all of the platelet-bound radioactivity becomes incorporated into clots formed by adding thrombin to whole blood. 7E3 was labeled with "'In by the cyclic anhydride diethylenetriaminepentaacetic acid method or by radioiodination with 123I. At a ratio of 1:50 (anhydride:7E3) the specific activity ranged between 10 and 40 gCi/,ug (1 Ci = 37 GBq) without change in the antibody characteristics. In vivo studies in dogs were performed by preincubating for 1 hr the radiolabeled 7E3 with citrated blood or by directly injecting the radiolabeled 7E3 intravenously. Experimental thrombi were induced by transcatheter placement of copper coils into peripheral arteries and veins as well as in the superior vena cava and pulmonary artery. With y camera, visualization of venous and arterial thrombi as well as sites of intimal injury without visible thrombi, could be observed 1-1.5 hr after injection. There was no need for delayed imaging because of the fast clearance of radioactivity from the circulation nor was there need for blood pool subtraction. Two to 10-hr thrombi could be imaged but 48-hr thrombi were not detectable with this method. No change in platelet counts before and after the injection of labeled 7E3 nor increased bleeding tendency occurred. The advantages of this method are a shorter preimaging preparation time, faster visualization after injection, and no need for delayed imaging or subtraction techniques. For these reasons human investigations seem to be warranted.Significant difficulties remain in the diagnosis of thrombotic vascular disease, especially in defining the precise anatomic location and extent of the thrombus. The techniques involving imaging the thrombus with radiolabeled fibrinogen and imaging the vascular obstruction with various radiopharmaceuticals both lack sensitivity and specificity, especially in sites beyond the extremities (1-3). Imaging with radiolabeled platelets was developed to overcome the limitations of these techniques, but it has not gained widespread use in clinical diagnosis primarily because the labeling procedure is complex and time-consuming. Moreover, the low target/background ratios achieved necessitate either delayed imaging and/or background subtraction to obtain su...

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“…ABT-806 was transferred into 0.1 mol/L sodium bicarbonate (pH 8.2) by size-exclusion chromatography and ultrafiltration. A 10-fold molar excess of DTPA was added to the antibody and incubated for 1 hour (15). Unbound DTPA was removed by size-exclusion chromatography and ultrafiltration and the DTPA-antibody was transferred into 0.1 mol/L sodium acetate (pH 7.2).…”

Section: Dtpa Conjugationmentioning

confidence: 99%

Characterization of ABT-806, a Humanized Tumor-Specific Anti-EGFR Monoclonal Antibody

Reilly

Phillips

Buchanan

et al. 2015

Molecular Cancer Therapeutics

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Despite clinical efficacy, current approved agents targeting EGFR are associated with on-target toxicities as a consequence of disrupting normal EGFR function. MAb 806 is a novel EGFR antibody that selectively targets a tumor-selective epitope suggesting that a mAb 806-based therapeutic would retain antitumor activity without the on-target toxicities associated with EGFR inhibition. To enable clinical development, a humanized variant of mAb 806 designated ABT-806 was generated and is currently in phase 1 trials. We describe the characterization of binding and functional properties of ABT-806 compared with the clinically validated anti-EGFR antibody cetuximab. ABT-806 binds the mutant EGFRvIII with high affinity and, relative to cetuximab, exhibits increased potency against glioblastoma multiforme cell line and patient-derived xenografts expressing this form of the receptor. ABT-806 also inhibits the growth of squamous cell carcinoma xenograft models expressing high levels of wild-type EGFR, associated with inhibition of EGFR signaling, although higher doses of ABT-806 than cetuximab are required for similar activity. ABT-806 enhances in vivo potency of standard-of-care therapies used to treat glioblastoma multiforme and head and neck squamous cell carcinoma. An indiumlabeled version of ABT-806, [ 111 In]-ABT-806, used to investigate the relationship between dose and receptor occupancy, revealed greater receptor occupancy at lowers doses in an EGFRvIII-expressing model and significant uptake in an orthotopic model. Collectively, these results suggest that ABT-806 may have antitumor activity superior to cetuximab in EGFRvIIIexpressing tumors, and similar activity to cetuximab in tumors highly overexpressing wild-type EGFR with reduced toxicity.

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Radioactive Labeling of Antibody: A Simple and Efficient Method

Cited by 374 publications

References 7 publications

Molecular probes for extracellular adenosine receptors

Molecular probes for extracellular adenosine receptors

Thrombus radioimmunoscintigraphy: an approach using monoclonal antiplatelet antibody.

Characterization of ABT-806, a Humanized Tumor-Specific Anti-EGFR Monoclonal Antibody

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