FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

Abstract

Fibroblast growth factor receptor 1 (FGFR1) transmits signals through the plasma membrane regulating essential cellular processes like division, motility, metabolism, and death. Overexpression of FGFR1 is observed in numerous tumors and thus constitutes an attractive molecular target for selective cancer treatment. Targeted anti‐cancer therapies aim for the precise delivery of drugs into cancer cells, sparing the healthy ones and thus limiting unwanted side effects. One of the key steps in targeted drug delivery is receptor‐mediated endocytosis. Here, we show that the efficiency and the mechanism of FGFR1 internalization are governed by the spatial distribution of the receptor in the plasma membrane. Using engineered antibodies of different valency, we demonstrate that dimerization of FGFR1 with bivalent antibody triggers clathrin‐mediated endocytosis (CME) of the receptor. Clustering of FGFR1 into larger oligomers with tetravalent antibody stimulates fast and highly efficient uptake of the receptor that occurs via two distinct mechanisms: CME and dynamin‐dependent clathrin‐independent endocytic routes. Furthermore, we show that all endocytic pathways engaged in FGFR1 internalization do not require receptor activation. Our data provide novel insights into the mechanisms of intracellular trafficking of FGFR1 and constitute guidelines for development of highly internalizing antibody‐based drug carriers for targeted therapy of FGFR1‐overproducing cancers.

FGF1 Fusions with the Fc Fragment of IgG1 for the Assembly of GFPpolygons-Mediated Multivalent Complexes Recognizing FGFRs

“…Regulation of RTK distribution in the plasma membrane is one of strategies for fine-tuning RTK-dependent signaling [10]. We have previously reported that by using engineered oligomeric FGFR1 ligands it is possible to control activity, endocytosis and cellular level of the receptor [21,22]. Importantly, the architecture of the oligomeric ligands (binding site on the receptor, affinity, oligomeric state, oligomerization scaffold used) played an important role in determining the outcome of FGFR1 modulation [21,22].…”

“…We have previously reported that by using engineered oligomeric FGFR1 ligands it is possible to control activity, endocytosis and cellular level of the receptor [21,22]. Importantly, the architecture of the oligomeric ligands (binding site on the receptor, affinity, oligomeric state, oligomerization scaffold used) played an important role in determining the outcome of FGFR1 modulation [21,22]. Therefore, we have sought to develop novel strategies to generate multivalent FGFR1 ligands.…”

“…Shifting FGFR1 from monomer to dimer with a bivalent engineered antibody stimulates clathrin-mediated endocytosis and lysosomal degradation of FGFR1, shortening the receptor's exposure at the surface [19,20]. High-order oligomerization of FGFR1 with a tetravalent engineered antibody or with FGF1 oligomers further enhances the efficiency of receptor endocytosis and accelerates its lysosomal targeting [21]. Importantly, FGFR1 clustering alters the mechanisms of receptor internalization by activating clathrin-independent, dynamin-dependent endocytic routes [21].…”

“…High-order oligomerization of FGFR1 with a tetravalent engineered antibody or with FGF1 oligomers further enhances the efficiency of receptor endocytosis and accelerates its lysosomal targeting [21]. Importantly, FGFR1 clustering alters the mechanisms of receptor internalization by activating clathrin-independent, dynamin-dependent endocytic routes [21]. Thirdly, tetravalent and pentavalent FGF1 oligomers alter FGFR1 activity by promoting mitogenic signaling and inhibiting cell metabolic activity [22].…”

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

“…Elevated level of FGFR1 has been observed in various tumor types, like breast, lung, head or neck cancers [ 16 – 21 ]. Thus, FGFR1 is considered as an attractive tumor biomarker and several approaches for selective treatment of FGFR1 overproducing cancer cells have been developed, including cytotoxic conjugates with antibody fragments or natural ligands as targeting agents [ 22 – 25 ]. We have recently shown that the spatial distribution of FGFR1 at the plasma membrane determines efficiency and mechanism of receptor endocytosis [ 25 ].…”