Cargando…
Bivalent EGFR-Targeting DARPin-MMAE Conjugates
Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bi...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8909960/ https://www.ncbi.nlm.nih.gov/pubmed/35269611 http://dx.doi.org/10.3390/ijms23052468 |
_version_ | 1784666323950764032 |
---|---|
author | Karsten, Lennard Janson, Nils Le Joncour, Vadim Alam, Sarfaraz Müller, Benjamin Tanjore Ramanathan, Jayendrakishore Laakkonen, Pirjo Sewald, Norbert Müller, Kristian M. |
author_facet | Karsten, Lennard Janson, Nils Le Joncour, Vadim Alam, Sarfaraz Müller, Benjamin Tanjore Ramanathan, Jayendrakishore Laakkonen, Pirjo Sewald, Norbert Müller, Kristian M. |
author_sort | Karsten, Lennard |
collection | PubMed |
description | Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5). |
format | Online Article Text |
id | pubmed-8909960 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-89099602022-03-11 Bivalent EGFR-Targeting DARPin-MMAE Conjugates Karsten, Lennard Janson, Nils Le Joncour, Vadim Alam, Sarfaraz Müller, Benjamin Tanjore Ramanathan, Jayendrakishore Laakkonen, Pirjo Sewald, Norbert Müller, Kristian M. Int J Mol Sci Article Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5). MDPI 2022-02-23 /pmc/articles/PMC8909960/ /pubmed/35269611 http://dx.doi.org/10.3390/ijms23052468 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Karsten, Lennard Janson, Nils Le Joncour, Vadim Alam, Sarfaraz Müller, Benjamin Tanjore Ramanathan, Jayendrakishore Laakkonen, Pirjo Sewald, Norbert Müller, Kristian M. Bivalent EGFR-Targeting DARPin-MMAE Conjugates |
title | Bivalent EGFR-Targeting DARPin-MMAE Conjugates |
title_full | Bivalent EGFR-Targeting DARPin-MMAE Conjugates |
title_fullStr | Bivalent EGFR-Targeting DARPin-MMAE Conjugates |
title_full_unstemmed | Bivalent EGFR-Targeting DARPin-MMAE Conjugates |
title_short | Bivalent EGFR-Targeting DARPin-MMAE Conjugates |
title_sort | bivalent egfr-targeting darpin-mmae conjugates |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8909960/ https://www.ncbi.nlm.nih.gov/pubmed/35269611 http://dx.doi.org/10.3390/ijms23052468 |
work_keys_str_mv | AT karstenlennard bivalentegfrtargetingdarpinmmaeconjugates AT jansonnils bivalentegfrtargetingdarpinmmaeconjugates AT lejoncourvadim bivalentegfrtargetingdarpinmmaeconjugates AT alamsarfaraz bivalentegfrtargetingdarpinmmaeconjugates AT mullerbenjamin bivalentegfrtargetingdarpinmmaeconjugates AT tanjoreramanathanjayendrakishore bivalentegfrtargetingdarpinmmaeconjugates AT laakkonenpirjo bivalentegfrtargetingdarpinmmaeconjugates AT sewaldnorbert bivalentegfrtargetingdarpinmmaeconjugates AT mullerkristianm bivalentegfrtargetingdarpinmmaeconjugates |