Novel targeted therapy based on dual warhead conjugates against FGFR-dependent cancers

Effective cancer therapy is the most important goal in oncology. The progress in the field of antibody drug conjugates encouraged us to verify if the attachment of two different cytotoxins to a targeting protein will lead to a more efficient conjugate. The Project stems from our findings that fibroblast growth factor 2 (FGF2) can replace antibody as a targeting protein. FGF2 conjugated with monomethyl auristatin E (MMAE) can be internalized and specifically destroy cancer cells overexpressing FGF receptor 1.

We will produce FGF2 conjugates containing two extremely cytotoxic warheads: MMAE and α-amanitin (AMN). Simultaneous application of two drugs on the same carrier molecule is an alternative for conventional combination therapy. Both toxins act according to completely different mechanisms.MMAE blocks microtubule formation and AMN is RNA polymerase II and III inhibitor. Both warheads should act in concert in a single cancer cell. Furthermore, MMAE can be secreted from cancer cell and via so called 'bystander effect’ destroy neighboring cells, whereas AMN shows outstanding activity in cells expressing multi-drug resistant transporters. So, the probability to kill all cancer cells is much higher, compared to application of single cytostatics.

To develop conjugates we will apply a modular strategy for conjugation, with the use of thiol-maleimide conjugation and synthesis of PEGylated peptide with auristatin and amanitin attached to FGF2. These methods will allow us to produce homogenous conjugates of defined stoichiometry containing from 1 up to 3 molecules of each drug. Dual-warhead conjugates will be compared with two respective single warhead conjugates in biochemical and cell culture studies. The most promising dual conjugate will be tested in animal models, including FGFR-dependent xenographs and patient-derived tumor models.

The approach is highly original and addresses trends in anti-cancer drug development: specific delivery, high level of drug loading combined with defined stoichiometry and overall homogeneity of conjugate. Most importantly, our proposed targeted strategy will have not only the potential to efficiently kill tumor cells reducing the side effects on healthy cells, but also to limit their ability to develop resistance.

2021

During the first year of the project, we have established and optimized methods to study internalization and localization of exemplary FGF2 conjugate (a conjugate of FGF2 with PEGylated MMAE), as well as the cytotoxicity assessment for FGFR1-positive cells. We have used a panel of cell lines derived from lung and breast cancers that are reported in the literature to have increased FGFR levels, and did a systematic evaluation of cytotoxicity of MMAE alone and a model conjugate. We have established that the assay conditions and planned concentration ranges are suitable for FGF2-conjugate cytotoxicity assessment, and three FGFR1-positive and two FGFR1-negative cell lines were chosen for further studies. With regard to conjugate preparation, we have produced FGF2 variants suitable for conjugation with the cytotoxic drug/drugs, with high yield and high purity. We have also designed and then synthesized peptide carriers enabling the coupling of one or two different cytotoxic agents with the FGF2 proteins. To have a deeper insight into mechanisms of dual-warhead conjugate action, we are developing separate assays
to monitor toxicity of MMAE and amanitin, drugs used in dual-warhead conjugates – for MMAE an assay based on microtubule morphology visualized by fluorescence microscopy has been tested with success for pilot FGF2 conjugate (MKC5). Analogously, we are working on developing a read out for amanitin toxicity, ie. RNA polymerase inhibition.

2022

During this reporting period, we have obtained single- and double-warhead conjugates of FGF2 with two cytotoxic drugs, MMAE and betaamanitin. Despite the fact that the conjugates had to be redesigned to accommodate the change from alpha- to beta-amanitin, extensive optimization allowed to obtain the amounts suitable for detailed biophysical characterization and describing the conjugates cytotoxicity level together with its mechanism. The conjugates proved to be similarly stable to FGF2 alone and have unhindered FGFR1 receptor binding properties. The dual-warhead conjugate is internalized through the FGFR1 receptor and follows the classical endocytic pathway. Cell-based assays with the use of single- and double-warhead conjugates showed that a desired cytotoxicity is observed when FGFR-positive cells are treated, while FGFR-negative cell lines exhibit satisfactory lack of response. Obtained results enabled initiating in vivo studies withselected conjugate.

Determination of the maximum tolerated dose demonstrated that FGF2 conjugate is well-tolerated by mice. To describe the effect of FGF2-double-warhead conjugate on tumours of mice with cancer cells-derived xenografts an extensive study will be performed with adjusted conjugate dosage, once a mid-scale batch of conjugates is purified.

Publications:

1. Krzyscik MA, Zakrzewska M, Sørensen V, Øy GF, Brunheim S, Haugsten EM, Mælandsmo GM, Wiedlocha A, Otlewski J (2021); Fibroblast Growth Factor 2 Conjugated with Monomethyl Auristatin E Inhibits Tumor Growth in a Mouse Model; Biomacromolecules, 22 (10), 4169-4180
   DOI 10.1021/acs.biomac.1c00662
2. Szybowska P, Kostas M, Wesche J, Haugsten EM, Wiedlocha A ; Negative Regulation of FGFR (Fibroblast Growth Factor Receptor) Signaling; Cells. 2021; 10(6):1342. DOI 10.3390/cells10061342
3. Wiedlocha A, Haugsten EM, Zakrzewska M.; Roles of the FGF-FGFR Signaling System in Cancer Development and Inflammation ; Cells. 2021; 10(9):2231. DOI 10.3390/cells10092231
4. Wiedlocha A, Haugsten EM, Zakrzewska M. Roles of the FGF-FGFR Signaling System in Cancer Development and Inflammation. Cells. 2021 Aug 28;10(9):2231. doi: 10.3390/cells10092231. PMID: 34571880; PMCID: PMC8471549.
5. Karolina Jendryczko, Jakub Rzeszotko, Mateusz Adam Krzyscik, Anna Kocyła, Jakub Szymczyk, Jacek Otlewski, Anna Szlachcic; Conjugation via Maleimide–Thiol Chemistry Does Not Affect Targeting Properties of Cysteine-Containing Anti-FGFR1 Peptibodies; Molecular Pharmaceutics 2022 19 (5), 1422-1433 DOI: 10.1021/acs.molpharmaceut.1c00946
6. Mateusz A. Krzyscik, Łukasz Opaliński, Jakub Szymczyk, Jacek Otlewski; Cyclic and dimeric fibroblast growth factor 2 variants with high biomedical potential; Int J Biol Macromol. 2022 Oct 1:218:243-258. doi: 10.1016/j.ijbiomac.2022.07.105. Epub 2022 Jul 22.
7. Daria Nawrocka , Mateusz Adam Krzyscik † , Katarzyna Dominika Sluzalska and Jacek Otlewski (2023); Dual-Warhead Conjugate Based on Fibroblast Growth Factor 2 Dimer Loaded with a-Amanitin and Monomethyl Auristatin E Exhibits Superior Cytotoxicity towards Cancer Cells Overproducing Fibroblast Growth Factor Receptor 1 ; Int J Mol Sci. 2023 Jun 14;24(12):10143. DOI: 10.3390/ijms241210143
8. Mateusz A Krzyscik, Natalia Porębska, Łukasz Opaliński, Jacek Otlewski; Targeting HER2 and FGFR-positive cancer cells with a bispecific cytotoxic conjugate combining anti-HER2 Affibody and FGF2; Int J Biol Macromol. 2024 Jan;254(Pt 1):127657. doi: 10.1016/j.ijbiomac.2023.127657. Epub 2023 Oct 28.

Research team

Partners:

Contact person:

Monika Nahorska (PhD)
+48 71 375 26 08
monika@biotech.uni.wroc.pl

Project is funded by the „Research” program of the Norway Grants operated by the National Centre for Research and Development, project no: NOR/POL NOR/DUALDRUG/0058/2019-00

Project budget: 6 508 272.00 zł, including the budget for the University of Wrocław: 4 224 250 zł.

Project is carried out at: University of Wrocław, Oslo University Hospital, Pure Biologics S.A. Project leader: University of Wrocław.

Project duration: 1.10.2020- 30.09.2023

www.norwaygrants.org