Fig 2: B‐Fc and T‐Fc bind FGFR1 with high affinity. (A, B) SPR‐determined kinetic parameters of the interaction between B‐Fc and T‐Fc, and FGFR1, respectively. The extracellular region of FGFR1 was immobilized on SPR sensors and incubated with various concentrations of B‐Fc and T‐Fc. K D, k on, and k off values are presented. (C) BLI comparison of B‐Fc and T‐Fc interaction with FGFR1. The extracellular region of FGFR1 was immobilized on BLI sensors and incubated either with B‐Fc or T‐Fc. The association and dissociation profiles were measured. (D) Upper panel, B‐Fc and T‐Fc interaction with FGFR1 on model cells. U2OS‐R1 cells stably producing FGFR1 were incubated with B‐Fc or T‐Fc on ice to prevent internalization of receptor–antibody complexes, or briefly at room temperature. Nuclei were labeled with NucBlue Live; cells were washed, and fixed; and bound antibodies were visualized with Zenon AF‐488 using fluorescence microscopy. Scale bars represent 20 µm. Lower panel, quantification of T‐Fc and B‐Fc cell binding at room temperature performed using zen 2.3 software based on three independent experiments. The signal of B‐Fc was set to 100%, and average intensity of T‐Fc in relation to B‐Fc ±SD was shown. (E) BN‐PAGE analysis of FGFR1 complexes with engineered antibodies. FGFR1‐Fc (0.1 μm) was incubated with B‐Fc (0.2 μm, 1 μm) and T‐Fc (0.2 μm, 1 μm), and proteins were separated on 4–10% BN‐PAGE gels and detected by western blotting.

Fig 3: Characterization of anti-FGFR1 engineered antibodies of different valency. (A) The schematic representation of structures of tetravalent (T-Fc) and bivalent (B-Fc) anti-FGFR1 engineered antibodies. Fc region of IgG (CH2 and CH3 domains) is labeled in gray, and scFv proteins (VH and VL fusions) are marked in blue. Antibody regions recognizing epitopes within FGFR1 are marked in orange. (B) Expression and purification of T-Fc and B-Fc. Levels and purity of T-Fc at different stages of protein expression and purification process were monitored with SDS/PAGE and western blotting with antibodies recognizing Fc fragment. (C) B-Fc and T-Fc are specific toward FGFR1. The extracellular regions of FGFR1, VEGFR2, and PDGFR were immobilized on SPR sensors and tested for the interaction with B-Fc, T-Fc, and commercial anti-VEGFR2 and anti-PDGFR antibodies with SPR. (D) Selectivity tests of B-Fc and T-Fc against FGFRs. The extracellular regions of FGFR2, FGFR3, and FGFR4 were immobilized on SPR sensors and tested for the interaction with B-Fc, T-Fc, and FGF1 as a control using SPR. (E) Engineered antibodies bind the D1 domain of the receptor. The full-length extracellular domain of FGFR1 (D1-D2-D3), FGFR1 variant lacking the D1 domain (FGFR1 D2-D3), and recombinant D1 domain (FGFR1 GST-D1) were immobilized on SPR sensors and tested for interaction with B-Fc and T-Fc using SPR.

Fig 4: Genetic silencing of FGFR1 or deletion of FGF2 attenuates exosome secretion.A doxycycline-inducible lentiviral shRNA targeting FGFR1 was used to create a stable HS-5 cell line. The cells were then treated with doxycycline to induce FGFR1 silencing and compared to a GIPZ lentiviral control. (A) Silencing of FGFR1 expression is shown by immunoblot of cell lysates. ECVs from doxycycline-treated cells were analyzed by (B) immunoblot or (C) Virocyt Virus Counter. *p<0.05. (D) Bone marrow was isolated from Fgf2 +/+ and -/- mice and cultured ex vivo to grow adherent marrow stroma. Equal numbers of cells were then plated, CM collected for 72 hr, and then ultracentrifuged to collect ECVs. The ECVs were quantified by Virocyt. *p<0.05. (E) Equal number of cultured marrow cells from Fgf2 +/+ and -/- mice were plated and then ECVs collected by ultracentrifugation and analyzed by immunoblot.

Fig 5: Intrinsic AKT activation induces FGFR1 inhibition resistance in lung-cancer cells.a Western blot analysis comparing main signaling pathways between control (HCC15), resistant (NCI-H520 and NCI-H1703) and sensitive (DMS114, H1581 and LK2) lung-cancer cell lines after FGFR1 inhibition with AZD4547. b Cell viability assay comparing sensitivity profiles of the six lung-cancer cell lines indicated to FGFR1 inhibition with DMSO treatment as standard. c, d Knockdown of FGFR1 with small interfering RNA (siRNA, SI02224684) in different lung-cancer cell lines validated by western blot analysis after 48 h (c) and proliferation of cells was compared on day 6 (d). e, f Cell viability assay showing the effect of combining the AKT inhibitor AZD5363 with the FGFR1 inhibitor AZD4547 at different concentrations after 96 h incubation in NCI-H520 (e) and NCI-H1703 (f) SQCLC cell lines. g Combination index plot showing the synergetic effect of combining AZD4547 with AZD5363 in NCI-H520 and NCI-H1703 cell lines calculated using CompuSyn software based on the Chou–Talalay drug interaction algorithm: CI < 1, synergetic effect; CI = 1, additive effect; CI > 1, antagonistic effect. h Immunohistochemical staining of SQCLC patient tissue samples with negative, weak and strong phosphorylated AKT signals. i, j H-score quantification of pAKT signals in the SQCLC patients with FGFR1 amplification (i) and in the SQCLC patients with FGFR1 overexpression (j). Statistical analysis was performed with the chi-squared test: ns (p > 0.05), *(p < 0.05), **(p ≤ 0.01) and ***(p ≤ 0.001). Mean values are plotted; error bars represent standard deviation.