Modular self-assembly system for development of oligomeric, highly internalizing and potent cytotoxic conjugates targeting fibroblast growth factor receptors

• Published in: Journal of biomedical science 2021-10, Vol.28 (1), p.1-69, Article 69
• Main Authors: Poźniak, Marta, PorÄbska, Natalia, JastrzÄbski, Kamil, KrzyÅcik, Mateusz Adam, KuciÅska, Marika, Zarzycka, Weronika, Barbach, Agnieszka, Zakrzewska, MaÅgorzata, Otlewski, Jacek, MiÄczyÅska, Marta, OpaliÅski, Åukasz
• Format: Article
• Language: English
• Subjects: Antibiotics; Antibodies; Antimitotic agents; Antineoplastic agents; Binding; Biomarkers; Cancer; Cancer therapies; Care and treatment; Cells; Chromatography; Clustering; Conjugates; Cytotoxicity; E coli; Endocytosis; FGF/FGFR; Fibroblast growth factor 1; Fibroblast growth factor receptor 1; Fibroblast growth factor receptors; Fibroblast growth factors; Fibroblasts; Growth factor receptors; Growth factors; Health aspects; Internalization; Kinases; Ligands; Modular systems; Oligomerization; Oligomers; Plasmids; Protein expression; Proteins; Receptors; Recognition; Scientific equipment and supplies industry; Self-assembly; Spatial distribution; Streptavidin; Targeted therapy; Toxicity; Tumor markers; Tumors; Valency
• ISSN: 1423-0127; 1021-7770; 1423-0127
• DOI: 10.1186/s12929-021-00767-x

Background Overexpression of FGFR1 is observed in numerous tumors and therefore this receptor constitutes an attractive molecular target for selective cancer treatment with cytotoxic conjugates. The success of cancer therapy with cytotoxic conjugates largely relies on the precise recognition of a cancer-specific marker by a targeting molecule within the conjugate and its subsequent cellular internalization by receptor mediated endocytosis. We have recently demonstrated that efficiency and mechanism of FGFR1 internalization are governed by spatial distribution of the receptor in the plasma membrane, where clustering of FGFR1 into larger oligomers stimulated fast and highly efficient uptake of the receptor by simultaneous engagement of multiple endocytic routes. Based on these findings we aimed to develop a modular, self-assembly system for generation of oligomeric cytotoxic conjugates, capable of FGFR1 clustering, for targeting FGFR1-overproducing cancer cells. Methods Engineered FGF1 was used as FGFR1-recognition molecule and tailored for enhanced stability and site-specific attachment of the cytotoxic drug. Modified streptavidin, allowing for controlled oligomerization of FGF1 variant was used for self-assembly of well-defined FGF1 oligomers of different valency and oligomeric cytotoxic conjugate. Protein biochemistry methods were applied to obtain highly pure FGF1 oligomers and the oligomeric cytotoxic conjugate. Diverse biophysical, biochemical and cell biology tests were used to evaluate FGFR1 binding, internalization and the cytotoxicity of obtained oligomers. Results Developed multivalent FGF1 complexes are characterized by well-defined architecture, enhanced FGFR1 binding and improved cellular uptake. This successful strategy was applied to construct tetrameric cytotoxic conjugate targeting FGFR1-producing cancer cells. We have shown that enhanced affinity for the receptor and improved internalization result in a superior cytotoxicity of the tetrameric conjugate compared to the monomeric one. Conclusions Our data implicate that oligomerization of the targeting molecules constitutes an attractive strategy for improvement of the cytotoxicity of conjugates recognizing cancer-specific biomarkers. Importantly, the presented approach can be easily adapted for other tumor markers. Keywords: FGF/FGFR, Endocytosis, Cancer, Targeted therapy