Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)

Glioblastoma is the most frequent and aggressive brain tumor in adults. Patient survival has marginally improved during the twenty-first century, averaging 14.6 months. Principal limitations of current diagnostic methods and therapeutic strategies of glioblastoma are the belated translation of relev...

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Authors: Ruiz López, Eduardo, Jiménez Schuhmacher, Alberto
Format: master thesis
Status:Published version
Publication Date:2023
Country:España
Institution:Universidad de Zaragoza
Repository:Zaguán. Repositorio Digital de la Universidad de Zaragoza
OAI Identifier:oai:zaguan.unizar.es:160834
Online Access:http://zaguan.unizar.es/record/160834
Access Level:Open access
Keyword:neurociencias
oncología
biología molecular
neurobiología molecular
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oai_identifier_str oai:zaguan.unizar.es:160834
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)
title Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)
spellingShingle Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)
Ruiz López, Eduardo
neurociencias
oncología
biología molecular
neurobiología molecular
title_short Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)
title_full Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)
title_fullStr Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)
title_full_unstemmed Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)
title_sort Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)
dc.creator.none.fl_str_mv Ruiz López, Eduardo
Jiménez Schuhmacher, Alberto
author Ruiz López, Eduardo
author_facet Ruiz López, Eduardo
Jiménez Schuhmacher, Alberto
author_role author
author2 Jiménez Schuhmacher, Alberto
author2_role author
dc.subject.none.fl_str_mv neurociencias
oncología
biología molecular
neurobiología molecular
topic neurociencias
oncología
biología molecular
neurobiología molecular
description Glioblastoma is the most frequent and aggressive brain tumor in adults. Patient survival has marginally improved during the twenty-first century, averaging 14.6 months. Principal limitations of current diagnostic methods and therapeutic strategies of glioblastoma are the belated translation of relevant biomarkers into the clinical practice and the high molecular restrictive conditions imposed by the blood-brain barrier (BBB). New efforts towards a personalized medicine are emerging to complementarily assist traditional stereotactic brain biopsies which may not depict the high intratumoral heterogeneity of glioblastoma. Immunotargeted tools offered a new window for molecular imaging approaches diagnosing and treating glioblastoma. These non-invasive, quantitative and real-time imaging methods merge the high antigen selectivity of antibodies or their derivatives with the high quality performance of current imaging technologies, which may allow a more precise management of glioblastoma patients in future.<br />Performing agnostic bioinformatic analysis in different patient datasets, ATP Binding Cassette subfamily C member 3 (ABCC3) was identified as a candidate molecular target of glioblastoma. Expression of ABCC3 gene correlated with glioma tumor grade and a worse patient overall survival. Suitability of ABCC3 as a plausible biomarker of glioblastoma was studied by molecular profiling of newly established patient-derived glioblastoma cell lines. Expression of ABCC3 was elevated while compared with healthy control and positively correlated with CD44, and TNFRSF1A, its cluster counterparts of new gene expression signatures described in these cell lines. Experiments with established cell lines strengthen the role of ABCC3 in multidrug resistance (MDR) and cancer stemness properties of glioblastoma. Either the overexpression or the abrogation of the expression of ABCC3 interfered with resistance to temozolomide (TMZ), in O6-methylguanine-DNA methyltransferase (MGMT)-expressing cells. Conversely, chemotherapy with TMZ increased ABCC3 levels in glioblastoma cell lines. Further insights in the plausible regulation between ABCC3 and SOX2 were described. Besides, expression of ABCC3 simultaneously increased with that of cancer stemness biomarkers in cultured neurospheres (specially SOX2, OCT4, and NANOG). The relevance of ABCC3 in glioblastoma pathophysiology prompted the upcoming development of immunotargeted tools.<br />Nanobodies represent an innovative alternative to conventional full-length antibodies. Their lower molecular weight, higher BBB transcytotic crossing and tumor uptake, and faster body clearance make nanobodies attractive to improve monoclonal antibody-based tools. Following a peptide-based strategy, nanobodies targeting ABCC3 were isolated from a previously constructed glioblastoma-specific phage-display library. NbA42 and NbA213, two nanobodies targeting the N-terminus region of ABCC3 comprising amino acids 3-32 (e.g. equivalent to peptide ABCC3-A), demonstrated the highest immunoreactive potential to selectively detect ABCC3. Both nanobodies recognized ABCC3 in glioblastoma cell lines. Further experiments showed that NbA42 and NbA213 identified ABCC3 in glioblastoma tumors of subcutaneous mouse models, after systemic administration. In addition, both nanobodies accuratelly assessed ABCC3 expression in tumors of orthotopic glioblastoma murine models. NbA42 and NbA213 were suggested to traverse BBB by mechanisms of transcytosis. Their radiolabeled 68Ga-NOTA-NbA42 and 68Ga-NOTA-NbA213 derivatives conserved ABCC3 immunoreactivity, enabling future studies to implement glioblastoma immuno-Positron Emission Tomography (immuno-PET) based on these radiotracers. Synergies of both nanobodies with chemotherapeutic drugs treating glioblastoma introduced a plausible use of NbA42 and NbA213 as therapeutic agents by inhibiting ABCC3. Nevertheless, nanobody engineering using humanization strategies to eliminate their immunogenicity will be needed. NbA42 and NbA213 pave the way for a complementarily earlier and more accurate non-invasive theragnosis of glioblastoma patients.<br />
publishDate 2023
dc.date.none.fl_str_mv 2023
dc.type.none.fl_str_mv info:eu-repo/semantics/masterThesis
info:eu-repo/semantics/publishedVersion
format masterThesis
status_str publishedVersion
dc.identifier.none.fl_str_mv http://zaguan.unizar.es/record/160834
url http://zaguan.unizar.es/record/160834
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by-nc/3.0/es
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc/3.0/es
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universidad de Zaragoza, Prensas de la Universidad
publisher.none.fl_str_mv Universidad de Zaragoza, Prensas de la Universidad
dc.source.none.fl_str_mv reponame:Zaguán. Repositorio Digital de la Universidad de Zaragoza
instname:Universidad de Zaragoza
instname_str Universidad de Zaragoza
reponame_str Zaguán. Repositorio Digital de la Universidad de Zaragoza
collection Zaguán. Repositorio Digital de la Universidad de Zaragoza
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repository.mail.fl_str_mv
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spelling Development and characterization of nanobodies targeting ABCC3 for glioblastoma immunoguided applications. (Desarrollo y caracterización de nanoanticuerpos dirigidos frente a ABCC3 para aplicaciones inmuno-guiadas de glioblastoma)Ruiz López, EduardoJiménez Schuhmacher, Albertoneurocienciasoncologíabiología molecularneurobiología molecularGlioblastoma is the most frequent and aggressive brain tumor in adults. Patient survival has marginally improved during the twenty-first century, averaging 14.6 months. Principal limitations of current diagnostic methods and therapeutic strategies of glioblastoma are the belated translation of relevant biomarkers into the clinical practice and the high molecular restrictive conditions imposed by the blood-brain barrier (BBB). New efforts towards a personalized medicine are emerging to complementarily assist traditional stereotactic brain biopsies which may not depict the high intratumoral heterogeneity of glioblastoma. Immunotargeted tools offered a new window for molecular imaging approaches diagnosing and treating glioblastoma. These non-invasive, quantitative and real-time imaging methods merge the high antigen selectivity of antibodies or their derivatives with the high quality performance of current imaging technologies, which may allow a more precise management of glioblastoma patients in future.<br />Performing agnostic bioinformatic analysis in different patient datasets, ATP Binding Cassette subfamily C member 3 (ABCC3) was identified as a candidate molecular target of glioblastoma. Expression of ABCC3 gene correlated with glioma tumor grade and a worse patient overall survival. Suitability of ABCC3 as a plausible biomarker of glioblastoma was studied by molecular profiling of newly established patient-derived glioblastoma cell lines. Expression of ABCC3 was elevated while compared with healthy control and positively correlated with CD44, and TNFRSF1A, its cluster counterparts of new gene expression signatures described in these cell lines. Experiments with established cell lines strengthen the role of ABCC3 in multidrug resistance (MDR) and cancer stemness properties of glioblastoma. Either the overexpression or the abrogation of the expression of ABCC3 interfered with resistance to temozolomide (TMZ), in O6-methylguanine-DNA methyltransferase (MGMT)-expressing cells. Conversely, chemotherapy with TMZ increased ABCC3 levels in glioblastoma cell lines. Further insights in the plausible regulation between ABCC3 and SOX2 were described. Besides, expression of ABCC3 simultaneously increased with that of cancer stemness biomarkers in cultured neurospheres (specially SOX2, OCT4, and NANOG). The relevance of ABCC3 in glioblastoma pathophysiology prompted the upcoming development of immunotargeted tools.<br />Nanobodies represent an innovative alternative to conventional full-length antibodies. Their lower molecular weight, higher BBB transcytotic crossing and tumor uptake, and faster body clearance make nanobodies attractive to improve monoclonal antibody-based tools. Following a peptide-based strategy, nanobodies targeting ABCC3 were isolated from a previously constructed glioblastoma-specific phage-display library. NbA42 and NbA213, two nanobodies targeting the N-terminus region of ABCC3 comprising amino acids 3-32 (e.g. equivalent to peptide ABCC3-A), demonstrated the highest immunoreactive potential to selectively detect ABCC3. Both nanobodies recognized ABCC3 in glioblastoma cell lines. Further experiments showed that NbA42 and NbA213 identified ABCC3 in glioblastoma tumors of subcutaneous mouse models, after systemic administration. In addition, both nanobodies accuratelly assessed ABCC3 expression in tumors of orthotopic glioblastoma murine models. NbA42 and NbA213 were suggested to traverse BBB by mechanisms of transcytosis. Their radiolabeled 68Ga-NOTA-NbA42 and 68Ga-NOTA-NbA213 derivatives conserved ABCC3 immunoreactivity, enabling future studies to implement glioblastoma immuno-Positron Emission Tomography (immuno-PET) based on these radiotracers. Synergies of both nanobodies with chemotherapeutic drugs treating glioblastoma introduced a plausible use of NbA42 and NbA213 as therapeutic agents by inhibiting ABCC3. Nevertheless, nanobody engineering using humanization strategies to eliminate their immunogenicity will be needed. NbA42 and NbA213 pave the way for a complementarily earlier and more accurate non-invasive theragnosis of glioblastoma patients.<br /><br />Universidad de Zaragoza, Prensas de la Universidad2023info:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://zaguan.unizar.es/record/160834reponame:Zaguán. Repositorio Digital de la Universidad de Zaragozainstname:Universidad de ZaragozaIngléshttp://creativecommons.org/licenses/by-nc/3.0/esinfo:eu-repo/semantics/openAccessoai:zaguan.unizar.es:1608342026-05-29T13:59:51Z
score 15.811543

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