The fibroblast growth factor receptor family members, FGFR1-4, are frequently overexpressed in various solid tumors, including breast cancer and sarcomas. This overexpression highlights the potential of the family of FGFRs as promising targets for cancer therapy. However, conventional FGFR kinase inhibitors often encounter challenges such as limited efficacy or drug resistance. In this study, we pursue an alternative strategy by designing a conjugate of the FGFR ligand FGF1 with the radioisotope 161Tb, for targeted therapy in FGFR-overexpressing cancer cells. FGF1 was engineered (eFGF1) to incorporate a single cysteine at the C terminus for site-specific labeling with a DOTA chelator. eFGF1-DOTA was mixed with the radioisotope 161Tb under mild conditions, resulting in a labeling efficiency above 90%. The nonradioactive ligands were characterized by mass spectrometry, while radioligands were characterized by thin-layer chromatography. The targeting function of the radioligands was assessed through confocal microscopy, flow cytometry, and Western blot analysis, focusing on binding to cancer cells and the activation of downstream signaling pathways related to FGFR. When compared to MCF-7 and RD cell lines with low FGFR expression, eFGF1-DOTA-Tb[161Tb] radioligands demonstrated significantly higher accumulation in FGFR-overexpressing cell lines (MCF-7 FGFR1 and RMS559), leading to enhanced cytotoxicity. Besides radionuclides, eFGF1 can also deliver doxorubicin (DOX) into cancer cells. Considering these characteristics, eFGF1-DOTA-Tb[161Tb] and eFGF1-DOX emerge as promising candidates for FGFR-targeted cancer therapy, and further evaluation in vivo is warranted.

Centre for Cancer Cell Reprogramming Institute of Clinical Medicine Faculty of Medicine University of Oslo Montebello Oslo 0379 Norway

Centrum výzkumu Řež s r o Hlavní 130 Řež Husinec 250 68 Czech Republic

Department of Microbiology Oslo University Hospital Rikshospitalet Oslo 0424 Norway

Department of Molecular Medicine Institute of Basic Medical Sciences University of Oslo Oslo 0372 Norway

Department of Radiation Biology Institute for Cancer Research The Norwegian Radium Hospital Montebello Oslo 0379 Norway

Department of Tracer Technology Institute of Energy Technology Instituttveien 18 Kjeller 2007 Norway

Department of Tumor Biology Institute for Cancer Research The Norwegian Radium Hospital Oslo University Hospital Montebello Oslo 0379 Norway

ELIXIR Norway Department of Informatics University of Oslo Oslo 0316 Norway

Faculty of Nuclear Sciences and Physical Engineering Czech Technical University Prague Břehová 7 Prague 1 110 00 Czech Republic

Thor Medical Karenslyst allé 9C Oslo 0278 Norway

Zobrazit více v PubMed

Wesche J.; Haglund K.; Haugsten E. M. Fibroblast growth factors and their receptors in cancer. Biochem. J. 2011, 437 (2), 199–213. 10.1042/BJ20101603. PubMed DOI

Katoh M.; Loriot Y.; Brandi G.; Tavolari S.; Wainberg Z. A.; Katoh M. FGFR-targeted therapeutics: clinical activity, mechanisms of resistance and new directions. Nat. Rev. Clin. Oncol. 2024, 21 (4), 312–329. 10.1038/s41571-024-00869-z. PubMed DOI

Helsten T.; Elkin S.; Arthur E.; Tomson B. N.; Carter J.; Kurzrock R. The FGFR Landscape in Cancer: Analysis of 4,853 Tumors by Next-Generation Sequencing. Clin. Cancer Res. 2016, 22 (1), 259–267. 10.1158/1078-0432.CCR-14-3212. PubMed DOI

Sobhani N.; Ianza A.; D’Angelo A.; Roviello G.; Giudici F.; Bortul M.; Zanconati F.; Bottin C.; Generali D. Current Status of Fibroblast Growth Factor Receptor-Targeted Therapies in Breast Cancer. Cells 2018, 7 (7), 76.10.3390/cells7070076. PubMed DOI PMC

Goke F.; Bode M.; Franzen A.; Kirsten R.; Goltz D.; Goke A.; Sharma R.; Boehm D.; Vogel W.; Wagner P.; et al. Fibroblast growth factor receptor 1 amplification is a common event in squamous cell carcinoma of the head and neck. Mod. Pathol. 2013, 26 (10), 1298–1306. 10.1038/modpathol.2013.58. PubMed DOI

Taylor J. G. t.; Cheuk A. T.; Tsang P. S.; Chung J. Y.; Song Y. K.; Desai K.; Yu Y.; Chen Q. R.; Shah K.; Youngblood V.; et al. Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models. J. Clin. Invest. 2009, 119 (11), 3395–3407. 10.1172/JCI39703. PubMed DOI PMC

Loriot Y.; Necchi A.; Park S. H.; Garcia-Donas J.; Huddart R.; Burgess E.; Fleming M.; Rezazadeh A.; Mellado B.; Varlamov S.; et al. Erdafitinib in Locally Advanced or Metastatic Urothelial Carcinoma. N. Engl. J. Med. 2019, 381 (4), 338–348. 10.1056/NEJMoa1817323. PubMed DOI

Subbiah V.; Verstovsek S. Clinical development and management of adverse events associated with FGFR inhibitors. Cell Rep. Med. 2023, 4 (10), 101204.10.1016/j.xcrm.2023.101204. PubMed DOI PMC

Abou-Alfa G. K.; Sahai V.; Hollebecque A.; Vaccaro G.; Melisi D.; Al-Rajabi R.; Paulson A. S.; Borad M. J.; Gallinson D.; Murphy A. G.; et al. Pemigatinib for previously treated, locally advanced or metastatic cholangiocarcinoma: a multicentre, open-label, phase 2 study. Lancet Oncol 2020, 21 (5), 671–684. 10.1016/S1470-2045(20)30109-1. PubMed DOI PMC

Facchinetti F.; Hollebecque A.; Braye F.; Vasseur D.; Pradat Y.; Bahleda R.; Pobel C.; Bigot L.; Déas O.; Florez Arango J. D.; et al. Resistance to Selective FGFR Inhibitors in FGFR-Driven Urothelial Cancer. Cancer discovery 2023, 13 (9), 1998–2011. 10.1158/2159-8290.CD-22-1441. PubMed DOI PMC

Goyal L.; Saha S. K.; Liu L. Y.; Siravegna G.; Leshchiner I.; Ahronian L. G.; Lennerz J. K.; Vu P.; Deshpande V.; Kambadakone A.; et al. Polyclonal Secondary FGFR2Mutations Drive Acquired Resistance to FGFR Inhibition in Patients with FGFR2 Fusion-Positive Cholangiocarcinoma. Cancer discovery 2017, 7 (3), 252–263. 10.1158/2159-8290.CD-16-1000. PubMed DOI PMC

Babina I. S.; Turner N. C. Advances and challenges in targeting FGFR signalling in cancer. Nat. Rev. Cancer 2017, 17 (5), 318–332. 10.1038/nrc.2017.8. PubMed DOI

Borek A.; Sokolowska-Wedzina A.; Chodaczek G.; Otlewski J. Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers. PLoS One 2018, 13 (2), e019219410.1371/journal.pone.0192194. PubMed DOI PMC

Sommer A.; Kopitz C.; Schatz C. A.; Nising C. F.; Mahlert C.; Lerchen H. G.; Stelte-Ludwig B.; Hammer S.; Greven S.; Schuhmacher J.; et al. Preclinical Efficacy of the Auristatin-Based Antibody-Drug Conjugate BAY 1187982 for the Treatment of FGFR2-Positive Solid Tumors. Cancer Res. 2016, 76 (21), 6331–6339. 10.1158/0008-5472.CAN-16-0180. PubMed DOI

Krzyscik M. A.; Zakrzewska M.; Sorensen V.; Oy G. F.; Brunheim S.; Haugsten E. M.; Maelandsmo G. M.; Wiedlocha A.; Otlewski J. Fibroblast Growth Factor 2 Conjugated with Monomethyl Auristatin E Inhibits Tumor Growth in a Mouse Model. Biomacromolecules 2021, 22 (10), 4169–4180. 10.1021/acs.biomac.1c00662. PubMed DOI PMC

Xie Y.; Su N.; Yang J.; Tan Q.; Huang S.; Jin M.; Ni Z.; Zhang B.; Zhang D.; Luo F.; et al. FGF/FGFR signaling in health and disease. Signal Transduction Targeted Ther. 2020, 5 (1), 181.10.1038/s41392-020-00222-7. PubMed DOI PMC

Sgouros G.; Bodei L.; McDevitt M. R.; Nedrow J. R. Radiopharmaceutical therapy in cancer: clinical advances and challenges. Nat. Rev. Drug Discovery 2020, 19 (9), 589–608. 10.1038/s41573-020-0073-9. PubMed DOI PMC

Cui X. Y.; Li Z.; Kong Z.; Liu Y.; Meng H.; Wen Z.; Wang C.; Chen J.; Xu M.; Li Y.; et al. Covalent targeted radioligands potentiate radionuclide therapy. Nature 2024, 630 (8015), 206–213. 10.1038/s41586-024-07461-6. PubMed DOI

Bodei L.; Herrmann K.; Schoder H.; Scott A. M.; Lewis J. S. Radiotheranostics in oncology: current challenges and emerging opportunities. Nat. Rev. Clin. Oncol. 2022, 19 (8), 534–550. 10.1038/s41571-022-00652-y. PubMed DOI PMC

Sartor O.; de Bono J.; Chi K. N.; Fizazi K.; Herrmann K.; Rahbar K.; Tagawa S. T.; Nordquist L. T.; Vaishampayan N.; El-Haddad G.; et al. Lutetium-177-PSMA-617 for Metastatic Castration-Resistant Prostate Cancer. N. Engl. J. Med. 2021, 385 (12), 1091–1103. 10.1056/NEJMoa2107322. PubMed DOI PMC

Milenic D. E.; Brady E. D.; Brechbiel M. W. Antibody-targeted radiation cancer therapy. Nat. Rev. Drug Discov 2004, 3 (6), 488–499. 10.1038/nrd1413. PubMed DOI

Hu B.; Liu T.; Li L.; Shi L.; Yao M.; Li C.; Ma X.; Zhu H.; Jia B.; Wang F. IgG-Binding Nanobody Capable of Prolonging Nanobody-Based Radiotracer Plasma Half-Life and Enhancing the Efficacy of Tumor-Targeted Radionuclide Therapy. Bioconjug Chem. 2022, 33 (7), 1328–1339. 10.1021/acs.bioconjchem.2c00209. PubMed DOI

Müller C.; Umbricht C. A.; Gracheva N.; Tschan V. J.; Pellegrini G.; Bernhardt P.; Zeevaart J. R.; Köster U.; Schibli R.; van der Meulen N. P. Terbium-161 for PSMA-targeted radionuclide therapy of prostate cancer. Eur. J. Nucl. Med. Mol. Imaging 2019, 46 (9), 1919–1930. 10.1007/s00259-019-04345-0. PubMed DOI PMC

Lehenberger S.; Barkhausen C.; Cohrs S.; Fischer E.; Grunberg J.; Hohn A.; Koster U.; Schibli R.; Turler A.; Zhernosekov K. The low-energy beta(−) and electron emitter (161)Tb as an alternative to (177)Lu for targeted radionuclide therapy. Nucl. Med. Biol. 2011, 38 (6), 917–924. 10.1016/j.nucmedbio.2011.02.007. PubMed DOI

Baum R. P.; Singh A.; Kulkarni H. R.; Bernhardt P.; Ryden T.; Schuchardt C.; Gracheva N.; Grundler P. V.; Koster U.; Muller D.; et al. First-in-Humans Application of (161)Tb: A Feasibility Study Using (161)Tb-DOTATOC. J. Nucl. Med. 2021, 62 (10), 1391–1397. 10.2967/jnumed.120.258376. PubMed DOI PMC

Zakrzewska M.; Krowarsch D.; Wiedlocha A.; Olsnes S.; Otlewski J. Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action. J. Mol. Biol. 2005, 352 (4), 860–875. 10.1016/j.jmb.2005.07.066. PubMed DOI

Zakrzewska M.; Krowarsch D.; Wiedlocha A.; Otlewski J. Design of fully active FGF-1 variants with increased stability. Protein Eng. Des Sel 2004, 17 (8), 603–611. 10.1093/protein/gzh076. PubMed DOI

Xia X.; Kumru O. S.; Blaber S. I.; Middaugh C. R.; Li L.; Ornitz D. M.; Sutherland M. A.; Tenorio C. A.; Blaber M. Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application. J. Pharm. Sci. 2016, 105 (4), 1444–1453. 10.1016/j.xphs.2016.02.010. PubMed DOI PMC

Wang Y.; Kilic O.; Rozumalski L.; Distefano M. D.; Wagner C. R. Targeted Drug Delivery by MMAE Farnesyl-Bioconjugated Multivalent Chemically Self-Assembled Nanorings Induces Potent Receptor-Dependent Immunogenic Cell Death. Bioconjug Chem. 2024, 35 (5), 582–592. 10.1021/acs.bioconjchem.4c00004. PubMed DOI PMC

Zakrzewska M.; Wiedlocha A.; Szlachcic A.; Krowarsch D.; Otlewski J.; Olsnes S. Increased Protein Stability of FGF1 Can. Compensate for Its Reduced Affinity for Heparin. J. Biol. Chem. 2009, 284 (37), 25388–25403. 10.1074/jbc.M109.001289. PubMed DOI PMC

Wiedlocha A.; Falnes P. O.; Madshus I. H.; Sandvig K.; Olsnes S. Dual mode of signal transduction by externally added acidic fibroblast growth factor. Cell 1994, 76 (6), 1039–1051. 10.1016/0092-8674(94)90381-6. PubMed DOI

Imamura T.; Engleka K.; Zhan X.; Tokita Y.; Forough R.; Roeder D.; Jackson A.; Maier J. A.; Hla T.; Maciag T. Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence. Science 1990, 249 (4976), 1567–1570. 10.1126/science.1699274. PubMed DOI

Zakrzewska M.; Zhen Y.; Wiedlocha A.; Olsnes S.; Wesche J. Size limitation in translocation of fibroblast growth factor 1 fusion proteins across the endosomal membrane. Biochemistry 2009, 48 (30), 7209–7218. 10.1021/bi9007353. PubMed DOI

Wesche J.; Malecki J.; Wiedlocha A.; Skjerpen C. S.; Claus P.; Olsnes S. FGF-1 and FGF-2 require the cytosolic chaperone Hsp90 for translocation into the cytosol and the cell nucleus. J. Biol. Chem. 2006, 281 (16), 11405–11412. 10.1074/jbc.M600477200. PubMed DOI

Malecki J.; Wiedlocha A.; Wesche J.; Olsnes S. Vesicle transmembrane potential is required for translocation to the cytosol of externally added FGF-1. EMBO J. 2002, 21 (17), 4480–4490. 10.1093/emboj/cdf472. PubMed DOI PMC

Sorensen V.; Zhen Y.; Zakrzewska M.; Haugsten E. M.; Walchli S.; Nilsen T.; Olsnes S.; Wiedlocha A. Phosphorylation of fibroblast growth factor (FGF) receptor 1 at Ser777 by p38 mitogen-activated protein kinase regulates translocation of exogenous FGF1 to the cytosol and nucleus. Mol. Cell. Biol. 2008, 28 (12), 4129–4141. 10.1128/MCB.02117-07. PubMed DOI PMC

Lö brich M.; Rief N.; Heckmann M.; Fleckenstein J.; Rübe C.; Uder M. In vivo formation and repair of DNA double-strand breaks after computed tomography examinations. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (25), 8989.10.1073/pnas.0501895102. PubMed DOI PMC

Culajay J. F.; Blaber S. I.; Archana K.; Blaber M. Thermodynamic-characterization-of-mutants-of-human-fibroblast-growth-factor-1-with-an-increased. Biochemistry 2000, 39 (24), 7158.10.1021/bi9927742. PubMed DOI

Junutula J. R.; Raab H.; Clark S.; Bhakta S.; Leipold D. D.; Weir S.; Chen Y.; Simpson M.; Tsai S. P.; Dennis M. S.; et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 2008, 26 (8), 925–932. 10.1038/nbt.1480. PubMed DOI

Lieser R. M.; Yur D.; Sullivan M. O.; Chen W. Site-Specific Bioconjugation Approaches for Enhanced Delivery of Protein Therapeutics and Protein Drug Carriers. Bioconjug Chem. 2020, 31 (10), 2272–2282. 10.1021/acs.bioconjchem.0c00456. PubMed DOI

Beck A.; Goetsch L.; Dumontet C.; Corvaia N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat. Rev. Drug Discov 2017, 16 (5), 315–337. 10.1038/nrd.2016.268. PubMed DOI

Wells J. A.; Kumru K. Extracellular targeted protein degradation: an emerging modality for drug discovery. Nat. Rev. Drug Discov 2024, 23 (2), 126–140. 10.1038/s41573-023-00833-z. PubMed DOI

Zakrzewska M.; Marcinkowska E.; Wiedlocha A. FGF-1: from biology through engineering to potential medical applications. Crit Rev. Clin Lab Sci. 2008, 45 (1), 91–135. 10.1080/10408360701713120. PubMed DOI

Haugsten E. M.; Sorensen V.; Brech A.; Olsnes S.; Wesche J. Different intracellular trafficking of FGF1 endocytosed by the four homologous FGF receptors. J. Cell Sci. 2005, 118 (17), 3869–3881. 10.1242/jcs.02509. PubMed DOI

Rivankar S. An overview of doxorubicin formulations in cancer therapy. J. Cancer Res. Ther 2014, 10 (4), 853–858. 10.4103/0973-1482.139267. PubMed DOI

Blaber S. I.; Culajay J. F.; Khurana A.; Blaber M. Reversible thermal denaturation of human FGF-1 induced by low concentrations of guanidine hydrochloride. Biophys. J. 1999, 77 (1), 470–477. 10.1016/S0006-3495(99)76904-3. PubMed DOI PMC

Abramson J.; Adler J.; Dunger J.; Evans R.; Green T.; Pritzel A.; Ronneberger O.; Willmore L.; Ballard A. J.; Bambrick J.; et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 2024, 630 (8016), 493–500. 10.1038/s41586-024-07487-w. PubMed DOI PMC

Tunyasuvunakool K.; Adler J.; Wu Z.; Green T.; Zielinski M.; Žídek A.; Bridgland A.; Cowie A.; Meyer C.; Laydon A.; et al. Highly accurate protein structure prediction for the human proteome. Nature 2021, 596 (7873), 590–596. 10.1038/s41586-021-03828-1. PubMed DOI PMC

Jones D. T.; Cozzetto D. DISOPRED3: precise disordered region predictions with annotated protein-binding activity. Bioinformatics 2015, 31 (6), 857–863. 10.1093/bioinformatics/btu744. PubMed DOI PMC

Plotnikov A. N.; Hubbard S. R.; Schlessinger J.; Mohammadi M. Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand-receptor specificity. Cell 2000, 101 (4), 413–424. 10.1016/S0092-8674(00)80851-X. PubMed DOI