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A (99m)Tc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen
INTRODUCTION: Prostate-specific membrane antigen (PSMA) is an extensively studied antigen for imaging prostate cancer. We prepared a single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, incorporating a His-tag for labelling with (99m)Tc tr...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Lippincott Williams & Wilkins
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508854/ https://www.ncbi.nlm.nih.gov/pubmed/28598898 http://dx.doi.org/10.1097/MNM.0000000000000698 |
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author | Nawaz, Saima Mullen, Gregory E.D. Blower, Philip J. Ballinger, James R. |
author_facet | Nawaz, Saima Mullen, Gregory E.D. Blower, Philip J. Ballinger, James R. |
author_sort | Nawaz, Saima |
collection | PubMed |
description | INTRODUCTION: Prostate-specific membrane antigen (PSMA) is an extensively studied antigen for imaging prostate cancer. We prepared a single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, incorporating a His-tag for labelling with (99m)Tc tricarbonyl, and evaluated its binding using human PCa cell lines. METHODS: J591(scFv) was expressed in HEK-293T cells and purified by metal ion affinity chromatography, followed by size exclusion chromatography. Stability and monomer/dimer ratios of purified scFv under different storage conditions were analysed by SDS-PAGE and analytical size exclusion chromatography. J591(scFv) was labelled with [Image: see text] at 37°C for 60 min. The stability of (99m)Tc-scFv in human serum was analysed by SDS-PAGE with autoradiography. Cell-binding studies were carried out using PC3LN3 (PSMA negative) and PC3LN3-PSMA (a variant engineered to express PSMA) cell lines. RESULTS: J591(scFv) was most stable to dimerisation on storage at −80°C compared with −20 and 4°C. Radiochemical yields of 85–90% were obtained with the final radiochemical purity of more than 99% after purification by gel filtration. In these small-scale studies, the maximum specific activity achieved was 7 MBq/μg. Liquid chromatography–mass spectrometry showed the formation of (99m)Tc-J591(scFv), which was radiochemically stable in serum, with no dissociation of (99m)Tc over 24 h. Cell-binding assays showed specific binding to PSMA-positive cells. CONCLUSION: J591(scFv) can be radiolabelled with [Image: see text] conveniently and efficiently. The labelled product was stable in serum. It showed selective binding to PSMA-positive cells compared with PSMA-negative cells. This potential radiotracer warrants evaluation in PCa xenograft models. |
format | Online Article Text |
id | pubmed-5508854 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Lippincott Williams & Wilkins |
record_format | MEDLINE/PubMed |
spelling | pubmed-55088542017-07-31 A (99m)Tc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen Nawaz, Saima Mullen, Gregory E.D. Blower, Philip J. Ballinger, James R. Nucl Med Commun Original Articles INTRODUCTION: Prostate-specific membrane antigen (PSMA) is an extensively studied antigen for imaging prostate cancer. We prepared a single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, incorporating a His-tag for labelling with (99m)Tc tricarbonyl, and evaluated its binding using human PCa cell lines. METHODS: J591(scFv) was expressed in HEK-293T cells and purified by metal ion affinity chromatography, followed by size exclusion chromatography. Stability and monomer/dimer ratios of purified scFv under different storage conditions were analysed by SDS-PAGE and analytical size exclusion chromatography. J591(scFv) was labelled with [Image: see text] at 37°C for 60 min. The stability of (99m)Tc-scFv in human serum was analysed by SDS-PAGE with autoradiography. Cell-binding studies were carried out using PC3LN3 (PSMA negative) and PC3LN3-PSMA (a variant engineered to express PSMA) cell lines. RESULTS: J591(scFv) was most stable to dimerisation on storage at −80°C compared with −20 and 4°C. Radiochemical yields of 85–90% were obtained with the final radiochemical purity of more than 99% after purification by gel filtration. In these small-scale studies, the maximum specific activity achieved was 7 MBq/μg. Liquid chromatography–mass spectrometry showed the formation of (99m)Tc-J591(scFv), which was radiochemically stable in serum, with no dissociation of (99m)Tc over 24 h. Cell-binding assays showed specific binding to PSMA-positive cells. CONCLUSION: J591(scFv) can be radiolabelled with [Image: see text] conveniently and efficiently. The labelled product was stable in serum. It showed selective binding to PSMA-positive cells compared with PSMA-negative cells. This potential radiotracer warrants evaluation in PCa xenograft models. Lippincott Williams & Wilkins 2017-08 2017-05-31 /pmc/articles/PMC5508854/ /pubmed/28598898 http://dx.doi.org/10.1097/MNM.0000000000000698 Text en Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CreativeCommonsAttributionLicense4.0) (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Original Articles Nawaz, Saima Mullen, Gregory E.D. Blower, Philip J. Ballinger, James R. A (99m)Tc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen |
title | A (99m)Tc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen |
title_full | A (99m)Tc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen |
title_fullStr | A (99m)Tc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen |
title_full_unstemmed | A (99m)Tc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen |
title_short | A (99m)Tc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen |
title_sort | (99m)tc-labelled scfv antibody fragment that binds to prostate-specific membrane antigen |
topic | Original Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508854/ https://www.ncbi.nlm.nih.gov/pubmed/28598898 http://dx.doi.org/10.1097/MNM.0000000000000698 |
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