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

Abstract

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.

Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers

“…The trimeric 3xGFPp_FGF1E_LPET_MMAE conjugate displays high cytotoxicity against FGFR1-producing cells while remaining neutral toward FGFR1-negative cells. Importantly, its cytotoxicity is one of the highest (EC 50 in the subnanomolar range) among the conjugates targeting FGFR1 described to date. ,,, For comparison, monomeric conjugates composed of FGF1 and MMAE display much higher EC 50 values (50–150 nM, depending on the cell line). , These data suggest that the oligomerization of the targeting molecules with GFPp scaffolds not only improves the selective delivery of cytotoxic drugs to cancer cells but also allows for monitoring the distribution and intracellular trafficking of the conjugate.…”

“… 25 , 26 , 28 , 53 For comparison, monomeric conjugates composed of FGF1 and MMAE display much higher EC 50 values (50–150 nM, depending on the cell line). 25 , 52 These data suggest that the oligomerization of the targeting molecules with GFPp scaffolds not only improves the selective delivery of cytotoxic drugs to cancer cells but also allows for monitoring the distribution and intracellular trafficking of the conjugate.…”

“… 30 , 52 Inspired by these findings, we have recently demonstrated that the streptavidin-based controlled oligomerization of cytotoxic conjugates targeting FGFR1 and HER2 receptors improves their cellular uptake and cytotoxicity. 52 …”

“… 13 − 16 We took advantage of our recent findings, demonstrating that FGFR1 clustering either with multivalent antibodies or oligomeric ligands enhances the efficiency and alters the mechanisms of FGFR1-mediated endocytosis. 30 − 32 , 52 Additionally, we have shown that oligomerization of FGF1, a natural FGFR1 ligand, constitutes an attractive tool to increase its affinity for the receptor. 30 , 52 Inspired by these findings, we have recently demonstrated that the streptavidin-based controlled oligomerization of cytotoxic conjugates targeting FGFR1 and HER2 receptors improves their cellular uptake and cytotoxicity.…”

“…Since the efficacy of the targeted therapy with cytotoxic conjugates largely depends on the properties of the targeting molecules, our aim was to develop a highly stable, high-affinity, and efficiently internalizing targeting molecule with an intrinsic fluorescence allowing for its tracking. As FGFR1 is overexpressed in various types of cancer, we decided to engineer a targeting molecule specific for this receptor. − We took advantage of our recent findings, demonstrating that FGFR1 clustering either with multivalent antibodies or oligomeric ligands enhances the efficiency and alters the mechanisms of FGFR1-mediated endocytosis. − , Additionally, we have shown that oligomerization of FGF1, a natural FGFR1 ligand, constitutes an attractive tool to increase its affinity for the receptor. , Inspired by these findings, we have recently demonstrated that the streptavidin-based controlled oligomerization of cytotoxic conjugates targeting FGFR1 and HER2 receptors improves their cellular uptake and cytotoxicity …”