Human Anti-FGFR3 Recombinant Antibody (clone PRO-001)

(A) RCJ cells overexpressing FGFR3 (RCJ-FGFR3) were serum starved, and then stimulated (+) with 20 ng/mL FGF9 for 5 minutes alone or following preincubation with a control (C) or a neutralizing anti-FGFR3 Fab (001). Total cell lysates were probed by Western blot with anti-P-JNK. In parallel, lysates were immunoprecipitated with anti-FGFR3 antibody and immunocomplexes were analyzed by Western with antiphosphotyrosine (4G10) and anti-FGFR3. (B) FDCP cells that stably express FGFR1, FGFR2, or FGFR3 were cultured in the presence of increasing amounts of PRO-001. Two days later, cell proliferation was determined by XTT analysis. Data are the average of duplicate cultures.

Trudel, S., Stewart, A. K., Rom, E., Wei, E., Li, Z. H., Kotzer, S.,... & Yayon, A. (2006). The inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t (4; 14) multiple myeloma cells. Blood, 107(10), 4039-4046.

PRO-001 binding to FGFR3 was determined by flow cytometric analysis using a PE-conjugated anti-human secondary antibody. (A) Competition of PRO-001 binding to cell-surface FGFR3 on B9ᵂᵀ cells. The filled histogram indicates parental B9 cells; dotted gray line, B9ᵂᵀ without FGF9; solid line, B9ᵂᵀ in the presence of FGF9. (B) PRO-001 binding to cell-surface WT and mutant FGFR3 on B9 cells. The filled histogram indicates parental cells; thick light gray line, B9Y373C; thick dark gray line, B9K650E; dotted gray line, B9G384D; dotted black line, B9WT; solid black line, B9807C. (C) B9 cells were stimulated with FGF and incubated with increasing concentrations of PRO-001 or 100 nM PD173074 for 48 hours, and cell viability was assessed by MTT-based assay. □ indicates unstimulated; ▪, FGF stimulated; ▧, FGF plus 1 μg/mL PRO-001; Embedded Image, FGF plus 3 μg/mL PRO-001; ▨, FGF plus 5 μg/mL PRO-001; ▨, FGF9 plus 100 nM PD173074. Proliferation index (PI) = (ODd2)/(ODd0), where ODd2 and ODd0 are the optical density (OD) at 48 hours and day 0, respectively. When PI = 1, cells did not proliferate after 48 hours in culture. Values represent mean ± SD of 4 independent experiments.

Trudel, S., Stewart, A. K., Rom, E., Wei, E., Li, Z. H., Kotzer, S.,... & Yayon, A. (2006). The inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t (4; 14) multiple myeloma cells. Blood, 107(10), 4039-4046.

(A) Human myeloma cell lines (HMCLs) expressing FGFR3 (UTMC2, H929, KMS11, KMS18) or not (8226) were stimulated with FGF and incubated with 5 μg/mL PRO-001, control antibody, or 100 nM PD173074. Growth at 48 hours was assessed by MTT assay and is reported as PI = (OD+FGF±DRUG)/(OD-FGF), where OD+FGF±DRUG is the OD in the presence of FGF with or without inhibitor and OD-FGF is the OD in the absence of FGF. When PI = 1, this indicates no enhanced growth with the addition of FGF. ▪ indicates stimulated with FGF; ▧, FGF plus control antibody;■, FGF plus 5 μg/mL PRO-001;, FGF plus 100 nM PD173074. (B) Flow cytometric analyses of ERK1/2 phosphorylation. UTMC2 cells were stimulated with aFGF (black line) or pretreated with 5 μg/mL PRO-001 and then stimulated with FGF9 (gray line). The filled histogram indicates unstimulated cells. (C) UTMC2 cells were cultured in media containing 2.5% FCS and 10 ng/mL FGF9 with control antibody (□), 5 μg/mL PRO-001 (■), or 100 nM PD173074 (▧) in the presence or absence of 50 ng/ml IL-6 or 50 ng/ml IGF-I or both. Cell viability after 48 hours was assessed by MTT assay and is reported as PI = (OD+CYTOKINES)/(OD-FGF), where OD+CYTOKINES is the OD in the presence of FGF with or without IL-6 or IGF-I or both and OD-FGF is the OD in the absence of FGF. When PI = 1, this indicates no enhanced growth with the addition of cytokines. (D) BMSCs alone or BMSCs together with UTMC2 cells (□) were cultured with control antibody (■) or 5 μg/mL PRO-001 (▧) for 72 hours and apoptosis was assessed by means of flow cytometric assay of annexin V binding and propidium iodide exclusion.

Trudel, S., Stewart, A. K., Rom, E., Wei, E., Li, Z. H., Kotzer, S.,... & Yayon, A. (2006). The inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t (4; 14) multiple myeloma cells. Blood, 107(10), 4039-4046.

(A) FDCP-FGFR3ˢ²⁹⁴ᶜ cells were cultured in the presence of increasing amounts of PRO-001 or control antibody (C). Two days later, cell proliferation was determined by XTT analysis. Data are the average of duplicate cultures. (B) Nude mice (3 in each group) were injected subcutaneously at 2 locations, one on each flank (a, right flank; b, left flank), with 2 × 10⁶ FDCP-FGFR3ˢ²⁹⁴ᶜ cells each. A week later, mice were randomized to receive PRO-001 or PBS control by intraperitoneal injection, according to the schedule described in Table 1. Tumor volume was estimated from measurements in 3 dimensions at 22 or 29 days after cell injection.

Trudel, S., Stewart, A. K., Rom, E., Wei, E., Li, Z. H., Kotzer, S.,... & Yayon, A. (2006). The inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t (4; 14) multiple myeloma cells. Blood, 107(10), 4039-4046.

(A) Freshly isolated BMNCs were stained with PRO-001 (filled) or control antibody (open) and then stained with PE-conjugated anti-human secondary antibody. Myeloma cells were identified by CD138 labeling. (B) Primary myeloma cells were incubated in the absence (filled) or presence of FGF (gray line) or preincubated with 5 μg/mL PRO-001 (black line) for 2 hours and then stimulated with FGF. ERK1/2 phosphorylation was assessed by flow cytometric analysis. (C) Primary myeloma cells were cultured in the presence of control Fab (left panel) or 5 μg/mL PRO-001 (right panel). Cells were harvested after 7 days and stained with annexin V-FITC and analyzed by flow cytometry. Myelomas cells were identified as CD138⁺. The total percentage of CD138⁺ cells is shown in the top right quadrant.

Trudel, S., Stewart, A. K., Rom, E., Wei, E., Li, Z. H., Kotzer, S.,... & Yayon, A. (2006). The inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t (4; 14) multiple myeloma cells. Blood, 107(10), 4039-4046.