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Nimotuzumab Site-Specifically Labeled with (89)Zr and (225)Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers

SIMPLE SUMMARY: Monoclonal antibodies (IgG) are excellent probes for targeting cell surface receptors for imaging and therapeutic applications. These theranostic agents are often developed by randomly conjugating radioisotopes/drugs/chelators to the primary amine of lysine or the sulfhydryl groups o...

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Autores principales: Solomon, Viswas Raja, Barreto, Kris, Bernhard, Wendy, Alizadeh, Elahe, Causey, Patrick, Perron, Randy, Gendron, Denise, Alam, Md. Kausar, Carr, Adriana, Geyer, C. Ronald, Fonge, Humphrey
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699480/
https://www.ncbi.nlm.nih.gov/pubmed/33233524
http://dx.doi.org/10.3390/cancers12113449
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author Solomon, Viswas Raja
Barreto, Kris
Bernhard, Wendy
Alizadeh, Elahe
Causey, Patrick
Perron, Randy
Gendron, Denise
Alam, Md. Kausar
Carr, Adriana
Geyer, C. Ronald
Fonge, Humphrey
author_facet Solomon, Viswas Raja
Barreto, Kris
Bernhard, Wendy
Alizadeh, Elahe
Causey, Patrick
Perron, Randy
Gendron, Denise
Alam, Md. Kausar
Carr, Adriana
Geyer, C. Ronald
Fonge, Humphrey
author_sort Solomon, Viswas Raja
collection PubMed
description SIMPLE SUMMARY: Monoclonal antibodies (IgG) are excellent probes for targeting cell surface receptors for imaging and therapeutic applications. These theranostic agents are often developed by randomly conjugating radioisotopes/drugs/chelators to the primary amine of lysine or the sulfhydryl groups of cysteine on the antibody. Random conjugation often alters the properties of the antibody. We have site-specifically radiolabeled nimotuzumab an anti-epidermal growth factor receptor (EGFR) monoclonal antibody with 89Zr and 225Ac using SpyTag: ∆N-SpyCatcher for positron emission tomography (PET) imaging and alpha particle radiotherapy, and evaluated these agents in a model of EGFR-positive triple negative breast cancer (TNBC). Nimotuzumab-SpyTag-∆N-SpyCatcher constructs showed improved binding in vitro compared with randomly conjugated constructs. 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher specifically delineated EGFR-positive xenograft in vivo using microPET/CT imaging. Compared with control treatment groups, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher more than doubled the survival of mice bearing EGFR-positive MDA-MB-231 TNBC xenograft. This work highlights a facile method to site-specifically radiolabel antibodies using SpyTag: ∆N-SpyCatcher. ABSTRACT: To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH(2)) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH(2) or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with (89)Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 ((225)Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of (89)Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC(50) of (225)Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and (225)Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, (225)Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to (225)Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. (225)Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.
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spelling pubmed-76994802020-11-29 Nimotuzumab Site-Specifically Labeled with (89)Zr and (225)Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers Solomon, Viswas Raja Barreto, Kris Bernhard, Wendy Alizadeh, Elahe Causey, Patrick Perron, Randy Gendron, Denise Alam, Md. Kausar Carr, Adriana Geyer, C. Ronald Fonge, Humphrey Cancers (Basel) Article SIMPLE SUMMARY: Monoclonal antibodies (IgG) are excellent probes for targeting cell surface receptors for imaging and therapeutic applications. These theranostic agents are often developed by randomly conjugating radioisotopes/drugs/chelators to the primary amine of lysine or the sulfhydryl groups of cysteine on the antibody. Random conjugation often alters the properties of the antibody. We have site-specifically radiolabeled nimotuzumab an anti-epidermal growth factor receptor (EGFR) monoclonal antibody with 89Zr and 225Ac using SpyTag: ∆N-SpyCatcher for positron emission tomography (PET) imaging and alpha particle radiotherapy, and evaluated these agents in a model of EGFR-positive triple negative breast cancer (TNBC). Nimotuzumab-SpyTag-∆N-SpyCatcher constructs showed improved binding in vitro compared with randomly conjugated constructs. 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher specifically delineated EGFR-positive xenograft in vivo using microPET/CT imaging. Compared with control treatment groups, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher more than doubled the survival of mice bearing EGFR-positive MDA-MB-231 TNBC xenograft. This work highlights a facile method to site-specifically radiolabel antibodies using SpyTag: ∆N-SpyCatcher. ABSTRACT: To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH(2)) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH(2) or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with (89)Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 ((225)Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of (89)Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC(50) of (225)Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and (225)Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, (225)Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to (225)Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. (225)Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model. MDPI 2020-11-20 /pmc/articles/PMC7699480/ /pubmed/33233524 http://dx.doi.org/10.3390/cancers12113449 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Solomon, Viswas Raja
Barreto, Kris
Bernhard, Wendy
Alizadeh, Elahe
Causey, Patrick
Perron, Randy
Gendron, Denise
Alam, Md. Kausar
Carr, Adriana
Geyer, C. Ronald
Fonge, Humphrey
Nimotuzumab Site-Specifically Labeled with (89)Zr and (225)Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers
title Nimotuzumab Site-Specifically Labeled with (89)Zr and (225)Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers
title_full Nimotuzumab Site-Specifically Labeled with (89)Zr and (225)Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers
title_fullStr Nimotuzumab Site-Specifically Labeled with (89)Zr and (225)Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers
title_full_unstemmed Nimotuzumab Site-Specifically Labeled with (89)Zr and (225)Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers
title_short Nimotuzumab Site-Specifically Labeled with (89)Zr and (225)Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers
title_sort nimotuzumab site-specifically labeled with (89)zr and (225)ac using spytag/spycatcher for pet imaging and alpha particle radioimmunotherapy of epidermal growth factor receptor positive cancers
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699480/
https://www.ncbi.nlm.nih.gov/pubmed/33233524
http://dx.doi.org/10.3390/cancers12113449
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