NeuroMab™ Anti-FGFR4 BBB Shuttle Antibody (NRZP-1022-ZP3898)

Figure 1. 1A-D LD1 binds FGFR4.

A, LD1 binds human (h), mouse (m), and macaque (c) FGFR4, but not hFGFR1, hFGFR2, or hFGFR3. Binding of LD1 to immobilized FGFR-Fc chimeric protein was determined by a solid-phase binding assay. B, Binding affinity of LD1 to mouse, cynomolgus monkey and human FGFR4 determined by surface plasmon resonance. C, Binding of LD1 to hFGFR4 expressed on the cell surface of stably transfected HEK293 cells measured by FACS (RFU=relative fluorescence units). D, Binding of LD1 to immobilized hFGFR4-Flag chimeric protein with point mutations as measured by solid-phase binding assay.

Figures 2A-2C LD1 inhibits FGFR4 activity.

A, LD1 inhibits the binding of FGFR4 to FGF1 and FGF19, as determined by solid-phase binding assays. B, LD1 inhibits FGF1-stimulated proliferation of BaF3 cells stably expressing FGFR4/R1. C, LD1 inhibits FGFR4 signaling in L6 cells stably expressing FGFR4.

Figure 3A-D LD1 inhibits FGFR4 bioactivity in hepatoma cell lines.

A, LD1 inhibits FGFR4 signaling in HEP3B cells as assessed by western blot. B, LD1 inhibits FGFR4-mediated CYP7α1 inhibition in HEP3B cells. CYP7α1 levels are expressed as fold expression relative to the level of untreated cells. C, LD1 inhibits FGFR4-regulated c-Fos expression in a panel of HCC cell lines. Results are expressed as fold expression relative to c-Fos levels in untreated cells. D, Inhibition of FGFR4 expression inhibits colony formation in JHH5 cells stably transfected with the FGFR4 shRNA doxycycline-inducible vector.

Figures 4A-C In vivo efficacy of LD1. A, LD1 inhibits FGF19-regulated c-Fos expression in mouse liver.

Results are expressed as fold expression relative to c-Fos levels in the liver of untreated mice. B, LD1 (30 mg/kg; twice weekly) inhibits the growth of HUH7 xenograft tumors in vivo. C, Effect of LD1 on FGFR4, CYP7α1, c-Fos and egr-1 mRNA expression in HUH-7 xenograft tumors.

Figure 5 LD1 inhibits the biological activity of FGFR4 in HUH7 cells. LD1 inhibits FGFR4-mediated CYP7α1 inhibition in HUH7 cells. CYP7α1 levels are expressed as fold expression relative to the level of untreated cells.

Figure 6 In vivo efficacy of LD1. LD1 (30 mg/kg) inhibits the growth of HUH7 xenograft tumors in vivo. The antitumor efficacy of LD1 was assessed on a bi-weekly basis.

Figure 7 Pharmacokinetics and distribution of anti-FGFR4 antibody variants.

(A) Comparison of chLD1 and hLD1.vB binding to FGFR4 using FGFR4 ELISA. (B) Comparison of day 16 tumor volumes of chLD1, hLD1.vB, and vehicle in a HUH7 human hepatocellular carcinoma xenograft model in CRL nu/nu mice. (C) Pharmacokinetics of chLD1 and hLD1.vBNCR nude mice dosed IV at 1 or 20 mg/kg and samples analyzed using FGFR4 ELISA. (D) Tissue distribution of 125I-chLD1 and 125I-hLD1.vB in NCR nude mice.

Figure 8 Identification of the interaction between hLD1.vB and mouse C3d.

(A) Detection of chLD1 (shaded bars) and hLD1.vB (white bars) after incubation in PBS/BSA or NCR nude mouse, rat, human, and cynomolgus monkey plasma for 48 hours. Percent recovery was determined using FGFR4 ELISA.

Figure 9 Identification of the interaction between hLD1.vB and mouse C3d.

(B) Plasma binding analysis of 125I-chLD1 (solid line) and 125I-hLD1.vB (dashed line). (C) Immunoprecipitation of mouse plasma.

Figure 10 In vivo efficacy of LD1.

E, DEN treatment with control antibody, LD1, or 1A6 (anti-FGF19 antibody) accelerated liver weight in FGF19-TG:FGFR4-WT mice.

Figure 11 LD1 inhibits the biological activity of FGFR4 in liver cancer cell lines.

F, Counts of colony formation of LD1-inhibited HCC cell lines. The values are expressed as a percentage of the number of colonies enumerated without the addition of LD1.

Figure 12 LD1 binds to FGFR4.

E, Binding of LD1 to hFGFR4-Flag chimeric protein with point mutations assessed by Western blot.