Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo

Abstract The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We prev...

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Autores principales:	Emanuel Peplau, Francesco De Rose, Andreas Eichinger, Sybille Reder, Markus Mittelhäuser, Giorgia Scafetta, Markus Schwaiger, Wolfgang A. Weber, Armando Bartolazzi, Calogero D’Alessandria, Arne Skerra
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f202779add984825b42f13aa3af80c55
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spelling	oai:doaj.org-article:f202779add984825b42f13aa3af80c552021-12-02T14:23:23ZEffective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo10.1038/s41598-021-86641-02045-2322https://doaj.org/article/f202779add984825b42f13aa3af80c552021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-86641-0https://doaj.org/toc/2045-2322Abstract The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin VH/VL regions of which one—employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66—showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (KD = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.Emanuel PeplauFrancesco De RoseAndreas EichingerSybille RederMarkus MittelhäuserGiorgia ScafettaMarkus SchwaigerWolfgang A. WeberArmando BartolazziCalogero D’AlessandriaArne SkerraNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-18 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Emanuel Peplau Francesco De Rose Andreas Eichinger Sybille Reder Markus Mittelhäuser Giorgia Scafetta Markus Schwaiger Wolfgang A. Weber Armando Bartolazzi Calogero D’Alessandria Arne Skerra Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo
description	Abstract The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin VH/VL regions of which one—employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66—showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (KD = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.
format	article
author	Emanuel Peplau Francesco De Rose Andreas Eichinger Sybille Reder Markus Mittelhäuser Giorgia Scafetta Markus Schwaiger Wolfgang A. Weber Armando Bartolazzi Calogero D’Alessandria Arne Skerra
author_facet	Emanuel Peplau Francesco De Rose Andreas Eichinger Sybille Reder Markus Mittelhäuser Giorgia Scafetta Markus Schwaiger Wolfgang A. Weber Armando Bartolazzi Calogero D’Alessandria Arne Skerra
author_sort	Emanuel Peplau
title	Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo
title_short	Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo
title_full	Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo
title_fullStr	Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo
title_full_unstemmed	Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo
title_sort	effective rational humanization of a pasylated anti-galectin-3 fab for the sensitive pet imaging of thyroid cancer in vivo
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/f202779add984825b42f13aa3af80c55
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Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo

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