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The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells
BACKGROUND: Antibody drug conjugates (ADCs) represent one of the most promising approaches in the current immuno-oncology research. The precise delivery of cytotoxic drugs to the cancer cells using ADCs specific for tumor-associated antigens enables sparing the healthy cells and thereby reduces unwa...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103757/ https://www.ncbi.nlm.nih.gov/pubmed/33962559 http://dx.doi.org/10.1186/s10020-021-00306-2 |
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author | Poźniak, Marta Porębska, Natalia Krzyścik, Mateusz Adam Sokołowska-Wędzina, Aleksandra Jastrzębski, Kamil Sochacka, Martyna Szymczyk, Jakub Zakrzewska, Małgorzata Otlewski, Jacek Opaliński, Łukasz |
author_facet | Poźniak, Marta Porębska, Natalia Krzyścik, Mateusz Adam Sokołowska-Wędzina, Aleksandra Jastrzębski, Kamil Sochacka, Martyna Szymczyk, Jakub Zakrzewska, Małgorzata Otlewski, Jacek Opaliński, Łukasz |
author_sort | Poźniak, Marta |
collection | PubMed |
description | BACKGROUND: Antibody drug conjugates (ADCs) represent one of the most promising approaches in the current immuno-oncology research. The precise delivery of cytotoxic drugs to the cancer cells using ADCs specific for tumor-associated antigens enables sparing the healthy cells and thereby reduces unwanted side effects. Overexpression of fibroblast growth factor receptor 1 (FGFR1) has been demonstrated in numerous tumors and thereby constitutes a convenient molecular target for selective cancer treatment. We have recently engineered tetravalent anti-FGFR1 antibody, T-Fc, and have demonstrated that it displays extremely efficient internalization into FGFR1 producing cells, a feature highly desirable in the ADC approach. We have revealed that T-Fc mediates clustering of FGFR1, largely enhancing the uptake of FGFR1-T-Fc complexes by induction of clathrin-independent endocytic routes. The aim of this study was to obtain highly internalizing cytotoxic conjugate of the T-Fc for specific delivery of drugs into FGFR1-positive cancer cells. METHODS: Conjugation of the T-Fc to a cytotoxic payload, vcMMAE, was carried out via maleimide chemistry, yielding the T-Fc-vcMMAE. The specific binding of the T-Fc-vcMMAE conjugate to FGFR1 was confirmed in vitro with BLI technique. Confocal microscopy and flow cytometry were applied to determine FGFR1-dependence of the T-Fc-vcMMAE internalization. Western blot analyses of FGFR1-dependent signaling were conducted to assess the impact of the T-Fc-vcMMAE on FGFR1 activation and initiation of downstream signaling cascades. Finally, using FGFR1-negative and FGFR1-possitive cell lines, the cytotoxic potential of the T-Fc-vcMMAE was evaluated. RESULTS: We have performed the efficient conjugation of the tetravalent engineered antibody with a cytotoxic drug and generated FGFR1-specific ADC molecule, T-Fc-vcMMAE. We have demonstrated that T-Fc-vcMMAE conjugate exhibits high selectivity and affinity for FGFR1, similarly to T-Fc. Furthermore, we have shown that T-Fc constitutes an effective drug delivery vehicle as T-Fc-vcMMAE was efficiently and selectively internalized by FGFR1-producing cells leading to their death. Interestingly, we show that the efficiency of the uptake of T-Fc-vcMMAE corresponds well with the cytotoxicity of the conjugate, but doesn’t correlate with the FGFR1expression level. CONCLUSION: Our results show that T-Fc-vcMMAE fulfills the key criteria for the successful cytotoxic drug carrier in a targeted approach against FGFR1-positive cancer cells. Furthermore, our data implicate that not solely expression level of the receptor, but rather its cellular trafficking should be taken into account for selection of suitable molecular targets and cancer models for successful ADC approach. |
format | Online Article Text |
id | pubmed-8103757 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-81037572021-05-07 The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells Poźniak, Marta Porębska, Natalia Krzyścik, Mateusz Adam Sokołowska-Wędzina, Aleksandra Jastrzębski, Kamil Sochacka, Martyna Szymczyk, Jakub Zakrzewska, Małgorzata Otlewski, Jacek Opaliński, Łukasz Mol Med Research Article BACKGROUND: Antibody drug conjugates (ADCs) represent one of the most promising approaches in the current immuno-oncology research. The precise delivery of cytotoxic drugs to the cancer cells using ADCs specific for tumor-associated antigens enables sparing the healthy cells and thereby reduces unwanted side effects. Overexpression of fibroblast growth factor receptor 1 (FGFR1) has been demonstrated in numerous tumors and thereby constitutes a convenient molecular target for selective cancer treatment. We have recently engineered tetravalent anti-FGFR1 antibody, T-Fc, and have demonstrated that it displays extremely efficient internalization into FGFR1 producing cells, a feature highly desirable in the ADC approach. We have revealed that T-Fc mediates clustering of FGFR1, largely enhancing the uptake of FGFR1-T-Fc complexes by induction of clathrin-independent endocytic routes. The aim of this study was to obtain highly internalizing cytotoxic conjugate of the T-Fc for specific delivery of drugs into FGFR1-positive cancer cells. METHODS: Conjugation of the T-Fc to a cytotoxic payload, vcMMAE, was carried out via maleimide chemistry, yielding the T-Fc-vcMMAE. The specific binding of the T-Fc-vcMMAE conjugate to FGFR1 was confirmed in vitro with BLI technique. Confocal microscopy and flow cytometry were applied to determine FGFR1-dependence of the T-Fc-vcMMAE internalization. Western blot analyses of FGFR1-dependent signaling were conducted to assess the impact of the T-Fc-vcMMAE on FGFR1 activation and initiation of downstream signaling cascades. Finally, using FGFR1-negative and FGFR1-possitive cell lines, the cytotoxic potential of the T-Fc-vcMMAE was evaluated. RESULTS: We have performed the efficient conjugation of the tetravalent engineered antibody with a cytotoxic drug and generated FGFR1-specific ADC molecule, T-Fc-vcMMAE. We have demonstrated that T-Fc-vcMMAE conjugate exhibits high selectivity and affinity for FGFR1, similarly to T-Fc. Furthermore, we have shown that T-Fc constitutes an effective drug delivery vehicle as T-Fc-vcMMAE was efficiently and selectively internalized by FGFR1-producing cells leading to their death. Interestingly, we show that the efficiency of the uptake of T-Fc-vcMMAE corresponds well with the cytotoxicity of the conjugate, but doesn’t correlate with the FGFR1expression level. CONCLUSION: Our results show that T-Fc-vcMMAE fulfills the key criteria for the successful cytotoxic drug carrier in a targeted approach against FGFR1-positive cancer cells. Furthermore, our data implicate that not solely expression level of the receptor, but rather its cellular trafficking should be taken into account for selection of suitable molecular targets and cancer models for successful ADC approach. BioMed Central 2021-05-07 /pmc/articles/PMC8103757/ /pubmed/33962559 http://dx.doi.org/10.1186/s10020-021-00306-2 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Research Article Poźniak, Marta Porębska, Natalia Krzyścik, Mateusz Adam Sokołowska-Wędzina, Aleksandra Jastrzębski, Kamil Sochacka, Martyna Szymczyk, Jakub Zakrzewska, Małgorzata Otlewski, Jacek Opaliński, Łukasz The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells |
title | The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells |
title_full | The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells |
title_fullStr | The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells |
title_full_unstemmed | The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells |
title_short | The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells |
title_sort | cytotoxic conjugate of highly internalizing tetravalent antibody for targeting fgfr1-overproducing cancer cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103757/ https://www.ncbi.nlm.nih.gov/pubmed/33962559 http://dx.doi.org/10.1186/s10020-021-00306-2 |
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