A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems

Denoël, Thibaut; Pedrelli, Luca; Pantaleo, Giuseppe; Prior, John O.

doi:10.3390/ph12040177

Cited by 9 publications

(7 citation statements)

References 28 publications

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“…In]In-DTPA-trastuzumab and [ 177 Lu]Lu-DOTA-trastuzumab53 and support in vivo applications.The immunoreactivity of [ 111 In]In-DTPA-trastuzumab is not affected by the radiolabeling procedureTo verify that [ 111 In]In-DTPA-trastuzumab retained its affinity to the HER2 antigen aer DTPA conjugation and radiolabeling, the immunoreactive fraction of the radioconjugate was quan-tied by cell-binding assays in human ovarian carcinoma SK-OV-3 (HER2-positive) and human breast adenocarcinoma MDA-MB-231 (HER2-negative) cells using an innite antigen excess 54,55. The binding specicity of[ 111 In]In-DTPA-trastuzumab to SK-OV-3 and MDA-MB-231 cells was measured aer incubating a constant amount of [ 111 In]In-DTPA-trastuzumab (15 ng mL À1 ) with increasing numbers (0.25-8 Â 10 6 ) of cells.…”

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confidence: 99%

Template directed synthesis of antibody Fc conjugates with concomitant ligand release

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Template directed synthesis of antibody Fc conjugates with concomitant ligand release

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“…The assumption was made of a single binding site per antigen. The mathematical aspect of this method was fully validated in a separate publication [36].…”

Section: Measurement Of Immunoreactivitymentioning

confidence: 99%

Preclinical Evaluation and Dosimetry of [111In]CHX-DTPA-scFv78-Fc Targeting Endosialin/Tumor Endothelial Marker 1 (TEM1)

et al. 2020

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Purpose: Endosialin/tumor endothelial marker-1 (TEM1) is an attractive theranostic target expressed by the microenvironment of a wide range of tumors, as well as by sarcoma and neuroblastoma cells. We report on the radiolabeling and preclinical evaluation of the scFv78-Fc, a fully human TEM1-targeting antibody fragment cross-reactive with mouse TEM1. Procedures: The scFv78-Fc was conjugated with the chelator p-SCN-Bn-CHX-A"-DTPA, followed by labeling with indium-111. The number of chelators per molecule was estimated by mass spectrometry. A conventional saturation assay, extrapolated to infinite antigen concentration, was used to determine the immunoreactive fraction of the radioimmunoconjugate. The radiopharmaceutical biodistribution was assessed in immunodeficient mice grafted with Ewing's sarcoma RD-ES and neuroblastoma SK-N-AS human TEM1-positive tumors. The full biodistribution studies were preceded by a dose-escalation experiment based on the simultaneous administration of the radiopharmaceutical with increasing amounts of unlabeled scFv78-Fc. Radiation dosimetry extrapolations to human adults were obtained from mouse biodistribution data according to established methodologies and additional assumptions concerning the impact of the tumor antigenic sink in the cross-species translation. Results: [ 111 In]CHX-DTPA-scFv78-Fc was obtained with a radiochemical purity 9 98 % after 1 h incubation at 42°C and ultrafiltration. It showed good stability in human serum and 9 70 % immunoreactive fraction. Biodistribution data acquired in tumor-bearing mice confirmed fast blood clearance and specific tumor targeting in both xenograft models. The radiopharmaceutical off-target uptake was predominantly abdominal. After a theoretical injection of [ 111 In]CHX-DTPA-scFv78-Fc to the reference person, the organs receiving the highest absorbed dose would be the spleen (0.876 mGy/MBq), the liver (0.570 mGy/MBq) and the kidneys (0.298 mGy/MBq). The Electronic supplementary material The online version of this article (

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“…However, the Lindmo method is dependent on experimental conditions and can sometimes lead to unreliable and/or overestimated results [26,27]. According to the recent study by Denoël et al [28], these limitations can be overcome by applying the rectangular hyperbola method of modern data analysis software (e.g., GraphPad Prism) to fit the binding curve of B/(B+S) as a function of cell concentration that provides the IRF at infinite antigen excess by extrapolation. The IRF of the [ 64 Cu]Cu-NODAGA-, [ 64 Cu]Cu-15-5-and [ 64 Cu]Cu-DOTA-immunoconjugates was, thus, determined on the HER2-positive human breast cancer cell line BT474 and compared with that obtained for trastuzumab radiolabelled with iodine-125 (see Table 1 and Figure S2 in Supplementary Materials).…”

Section: In Vitro Receptor Binding Assaysmentioning

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Synthesis and In Vitro Comparison of DOTA, NODAGA and 15-5 Macrocycles as Chelators for the 64Cu-Labelling of Immunoconjugates

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The development of 64Cu-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators (BFCs) that contain functional groups allowing both convenient bioconjugation and stable copper complexes to limit in vivo bioreduction, transmetallation and/or transchelation. The excellent in vivo kinetic inertness of the pentaazamacrocyclic [64Cu]Cu-15-5 complex prompted us to investigate its potential for the 64Cu-labelling of monoclonal antibodies (mAbs), compared with the well-known NODAGA and DOTA chelators. To this end, three NODAGA, DOTA and 15-5-derived BFCs, containing a pendant azadibenzocyclooctyne moiety, were synthesised and a robust methodology was determined to form covalent bonds between them and azide-functionalised trastuzumab, an anti-HER2 mAb, using strain-promoted azide-alkyne cycloaddition. Unlike the DOTA derivative, the NODAGA- and 15-5-mAb conjugates were radiolabelled with 64Cu, obtaining excellent radiochemical yields, under mild conditions. Although all the radioimmunoconjugates showed excellent stability in PBS or mouse serum, [64Cu]Cu-15-5- and [64Cu]Cu-NODAGA-trastuzumab presented higher resistance to transchelation when challenged by EDTA. Finally, the immunoreactive fraction of the radioimmunoconjugates (88–94%) was determined in HER-2 positive BT474 human breast cancer cells, confirming that the bioconjugation and radiolabelling processes implemented had no significant impact on antigen recognition.

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A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems

Cited by 9 publications

References 28 publications

Template directed synthesis of antibody Fc conjugates with concomitant ligand release

Template directed synthesis of antibody Fc conjugates with concomitant ligand release

Preclinical Evaluation and Dosimetry of [111In]CHX-DTPA-scFv78-Fc Targeting Endosialin/Tumor Endothelial Marker 1 (TEM1)

Synthesis and In Vitro Comparison of DOTA, NODAGA and 15-5 Macrocycles as Chelators for the 64Cu-Labelling of Immunoconjugates

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