Human Anti-FGFR2 Recombinant Antibody (clone M048-D01) (CAT#: NS-027CN)

Figure 1 Induction of phosphorylated FGFR2 (P-FGFR2) levels after short term (15 min) incubation with anti FGFR2 antibodies at 1 mg/ml in MFM223 cells. Y is "% of untreated control cells".

As shown antibodies M048-D01-hIgGl and M047-D08-hIgGl increase the ELISA signal of P-FGFR2 by a factor greater 4 fold compared with untreated control cells. In contrast neither the control IgG antibody nor anti FGFR2 antibodies showed any significant effect on P-FGFR2 levels after short-term incubation. These results reveal an agonistic effect of anti FGFR2 antibodies on FGFR2 after short-term incubation.

Figure 2 Desensitizing of MFM223 cells against FGF7 (25 ng/ml, 15min) mediated induction of P-FGFR2 levels after long term (24h) incubation with anti FGFR2 antibodies at 1 mg/ml. Y is "% f untreated control cells".

As shown the antibodies M048-D0I-hIgGl and M047-D08-hIgGl reduce the level of P-FGFR2 which can be achieved after FGF7 stimulation very pronounced. In cells treated without antibody treatment as well as in cells treated with isotype control IgG stimulation with FGF7 lead to an about 4 fold increase of P-FGFR2 levels. In contrast, in samples pretreated with anti FGFR2 antibodies for 24h, FGF7 only induced P-FGFR2 levels by 1.37-1.4 fold. Taken together these results show that prolonged incubation of cells with anti FGFR2 antibodies leads to desensitization towards stimulation with FGF7,

Figure 3 Downregulation of FGFR2 surface expression in cell lines with FGFR2 overexpression (MFM223, SNU16) or FGFR2 mutations (AN3-CA, MFE-296) 4.5 h after incubation with anti FGFR2 antibodies at mg/ml measured by FACS analysis. Y is "% of control ceils".

As shown antibodies M048-D01-hIgGl and M047-D08-hIgGl are the only antibodies that reduce FGFR2 surface expression with FGFR2 overexpressing cell lines (MFM223, SNU16) and cells lines having FGFR2 mutations (AN3-CA, MFE-296). Antibodies like MAB684 and MAB6843 only reduce FGFR2 surface expression with cell lines which do not overexpress FGFR2. Antibodies like GAL-FR21 do not reduce FGFR2 surface expression with cell lines having FGFR2 mutations.

Figure 4 Downregulation of total FGFR2 levels after long term (96h) incubation with anti FGFR2 antibodies in S U16 cells. Y is "% of control cells". X is "Antibody concentration [ mg/ml]".

As shown antibodies M048-D01-hIgGl (white) and M047-D08-hIgGl (striped) decrease the total FGFR2 levels significantly after 96h in a dose dependent manner. A non-binding control antibody (black) does not show any effects. These results indicate that anti FGFR2 antibodies M048-D01-hIgGl and M047-D08-hIgGl do not only lead to a short term decrease in surface FGFR2 levels but also a long term reduction of total FGFR2 levels.

Figure 5 Microscopic evaluation of the time course of specific internalization of M048-DOl-hlgGl and M047-D08-hIgGl upon binding to endogenous FGFR2 expressing cells.

Y is "granule counts per cell". X is "time [min]". Internalization of antibodies was investigated on breast cancer cell line SUM 52PE. The granule counts per cell were measured in a kinetic fashion. As shown antibodies M048-D01-hIgGl (black squares and solid line) and M047-D08-hIgGl (black triangles and dashed line) show a rapid internalization as indicated by increasing granule count per cell. An isotype control antibody (stars and dashed line) does not show any internalization.

Figure 6 Internalization of M048-D01-hIgG1 (A, B) and M047-D08-hIgG1 (C, D) in SUM 52PE cells showed co-staining as indicated with Rab 7 (A, C) and not with Rab 1 1 (B, D). Internalization of GAL-FR21 (E, F) and GAL-FR22 (G,H) in SUM 52PE cells showed co-staining as indicated with Rab 1 1 (F, I I) and not with Rab 7 (E, G).

Figure 7 Growth of subcutaneous SNU-16 xenografts under intraperitoneal treatment with 2 mg/kg of M048-D0I-hIgGl (open triangles, solid line) in comparison to PBS (filled circles, solid line) and control IgG treatment (filled triangles, solid line).

Mean + standard deviation are plotted. X is "time after tumor inoculation [days]". Y is "tumor area [mm2]". Treatment with M048-D0 I-hlgG I resulted in a very significant tumor growth inhibition.

Figure 8 Dot plots of the tumor area of subcutaneous 4T 1 tumors at day 13 after tumor cell inoculation, the last time point before tumors became necrotic.

At this time point mice recived treatment with PBS alone (A), 5 mg/kg of M048-D01-hIgGl twice weekly i.v. (B), 100 mg/kg Lapatinib p.o. (C) or with 5 mg/kg of M048-D01-hIgGl twice weekly i.v. and 100 mg/kg Lapatinib p.o. (D). Y is tumor area [mm²] at day 13, dotted lines indicate the mean values, solid lines indicate the medians. Treatment with M048-D01-higGl alone resulted in a significant reduction of tumor area, while Lapatinib alone did not significantly affect tumor area. Combination of M048-D01-hIgGl with Lapatinib resulted in a significantly additive anti-tumor activity.

Figure 9 Growth of subcutaneous patient-derived GC 10-0608 xenografts under intraperitoneal treatment with 5 mg/kg (filled triangles, solid line), 2 mg/kg (filled circles, dashed line) and 1 mg/kg (filled squares, dotted line) of M048-D01-hIgGl in comparison to PBS (open diamonds, solid line).

Mean ± standard error of the means are plotted. X is "time under treatment [days]". Y is "tumor volume [mm³]". Treatment with all three doses of M048-D01-hIgGl resulted in a significant tumor growth inhibition.

Figure 10 Downregulation of total FGFR2 [total FGFR2] and phosphorylated FGFR2 [P-FGFR2] after long term treatment of SNU16 xenografts with anti FGFR2 antibodies M048-D0-hIgGl and M047-D08-hIgGl in comparison with a control antibody (2mg/kg, twice weekly, i.p., samples were taken 24h after the last dose).

As shown after treatment with M048-D01-hIgGl and M047-D08-hIgGl total FGFR2 [total FGFR2] and phosphorylated FGFR2 [P-FGFR2] were reduced significantly in comparison with treatment with control IgGl. Actin served as loading control.

Figure 1 Human Anti-FGFR2 Recombinant Antibody (NS-027CN) in SDS-PAGE

SDS-PAGE analysis of NS-027CN in reduced (Lane 2) and non-reduced (Lane 1) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively.

The purity of NS-027CN was greater than 95% as determined by SDS-PAGE.

Figure 2 Human Anti-FGFR2 Recombinant Antibody (NS-027CN) in SEC-HPLC

The purity of NS-027CN was greater than 95% as determined by SEC-HPLC.

Figure 3 Human Anti-FGFR2 Recombinant Antibody (NS-027CN) in WB

Western blot analysis of NS-027CN was performed by loading Recombinant Human FGFR2 Protein (ECD, His Tag).
NS-027CN incubation concentration: 2 ng/μL.
The secondary antibody: HRP-Goat Anti-Human IgG (H+L)
Lane 1: Reducing Antigen (0.1 μg)
Lane 2: Reducing Antigen (0.3 μg)
Lane 3: Reducing Antigen (0.6 μg)

Figure 4 Human Anti-FGFR2 Recombinant Antibody (NS-027CN) in ELISA

ELISA analysis of NS-027CN was performed by coating with Recombinant Human FGFR2 Protein (ECD, His Tag).
The secondary antibody: HRP-Goat Anti-Human IgG (H+L)

Figure 5 Human Anti-FGFR2 Recombinant Antibody (NS-027CN) in Dot Blot

Dot Blot analysis of NS-027CN was performed by coating with Recombinant Human FGFR2 Protein (ECD, His Tag).
NS-027CN incubation concentration: 2 ng/μL.
The secondary antibody: HRP-Goat Anti-Human IgG (H+L)