High Affinity Promotes Internalization of Engineered Antibodies Targeting FGFR1.

Fibroblast growth factor receptor 1 (FGFR1) is a plasma membrane protein that transmits signals from the extracellular environment, regulating cell homeostasis and function. Dysregulation of FGFR1 leads to the development of human cancers and noncancerous diseases. Numerous tumors overproduce FGFR1, making this receptor a perspective target for cancer therapies. Antibody-drug conjugates (ADCs) are highly potent and selective anticancer agents. ADCs are composed of antibodies (targeting factors) fused to highly cytotoxic drugs (warheads). The efficiency of ADC strategy largely depends on the internalization of cytotoxic conjugate into cancer cells. Here, we have studied an interplay between affinity of anti-FGFR1 antibodies and efficiency of their cellular uptake. We have developed a unique set of engineered anti-FGFR1 antibodies that bind the same epitope in the extracellular part of FGFR1, but with different affinities. We have demonstrated that these antibodies are effectively taken up by cancer cells in the FGFR1-dependent manner. Interestingly, we have found that efficiency, defined as rate and level of antibody internalization, largely depends on the affinity of engineered antibodies towards FGFR1, as high affinity antibody displays fastest internalization kinetics. Our data may facilitate design of therapeutically relevant targeting molecules for selective treatment of FGFR1 overproducing cancers.

The autoinhibitory function of D1 domain of FGFR1 goes beyond the inhibition of ligand binding.

Fibroblast growth factors (FGFs) and their plasma membrane-localized receptors (FGFRs) transduce signals that regulate developmental processes and metabolism. In numerous cancer types genetic aberrations of FGFR1 lead to its uncontrolled activation. To circumvent the unrestrained signal transduction, several intramolecular inhibitory mechanisms within FGFR1 have evolved. In vitro experiments with receptor truncation have demonstrated that the N-terminal D1 domain of FGFR1 negatively regulates ligand binding to the receptor. Here, we show that D1-specific monovalent antibody fragments can activate FGFR1 and its downstream signaling cascades in the absence of ligand. These data suggest that the D1 domain of FGFR1 may play autoinhibitory role not only by controlling ligand binding, but also by regulating the overall conformation of FGFR1, keeping it in a state that disfavors autoactivation in the absence of its cognate growth factor.

Antibody-induced dimerization of FGFR1 promotes receptor endocytosis independently of its kinase activity.

Fibroblast growth factors (FGFs) and their plasma membrane-localized receptors (FGFRs) play a key role in the regulation of developmental processes and metabolism. Aberrant FGFR signaling is associated with the progression of serious metabolic diseases and human cancer. Binding of FGFs to FGFRs induces receptor dimerization and transphosphorylation of FGFR kinase domains that triggers activation of intracellular signaling pathways. Following activation, FGFRs undergo internalization and subsequent lysosomal degradation, which terminates transmission of signals. Although factors that regulate FGFR endocytosis are continuously discovered, little is known about the molecular mechanism that initiates the internalization of FGFRs. Here, we analyzed the internalization of antibody fragments in various formats that target FGFR1. We show that FGFR1-specific antibody fragments in the monovalent scFv format bind to FGFR1, but are not internalized into cells that overproduce FGFR1. In contrast, the same scFv proteins in the bivalent scFv-Fc format are efficiently internalized via FGFR1-mediated, clathrin and dynamin dependent endocytosis. Interestingly, the receptor tyrosine kinase activity is dispensable for endocytosis of scFv-Fc-FGFR1 complexes, suggesting that only dimerization of receptor is required to trigger endocytosis of FGFR1 complexes.