Human Anti-IGF1R Recombinant Antibody (clone m590) (CAT#: FAMAB-0608WJ)

Figure 1 Flow cytometry of breast cancer MCF-7 and ovarian cancer SKOV-3 cells stained with m590 and trastuzumab at 10 μg/mL.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 2 Simultaneous binding of Bi-Ab to recombinant IGF-IR and HER2 ectodomains by indirect ELISA.

Recombinant IGF-IR ectodomain was coated in microplates. Three-fold serially diluted Bi-Ab, trastuzumab, and m590 were added to the wells followed by addition of 100 ng per well of biotinylated recombinant HER2 ectodomain. Bound HER2 ectodomain was detected by HRP conjugated to streptavidin and TMB.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 3 Histogram overlay of SKOV-3 cells stained with m590, trastuzumab, and Bi-Ab at 2 and 10 μg/mL.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 4 Binding kinetics of Bi-Ab, m590, and trastuzumab to membrane-associated IGF-IR and HER2 on SKOV-3 cells.

Mean fluorescence intensity (MFI) of SKOV-3 cells stained with the antibodies at different concentrations was measured.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 5 A and B, inhibition of IGF-I (1.5 nmol/L) induced phosphorylation of Akt and ERK by m590 (40 nmol/L) alone (A) or in combination with trastuzumab (6.7 nmol/L; B) in MCF-7 cells.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 6 Inhibition of IGF-I (1.5 nmol/L) induced phosphorylation of IGF-IR in SKOV-3 cells.

All antibodies were tested at 100 μg/mL and incubated with cells for 24 hours.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 7 D and E, inhibition of IGF-I (1.5 nmol/L) induced phosphorylation of Akt and ERK in MCF-7 (D) and SKOV-3 (E) cells.

All antibodies were tested at 100 μg/mL and incubated with cells for 24 hours.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 8 Inhibition of phosphorylation of Akt and ERK by the antibodies in SKOV-3 cells in the absence of IGF-I.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 9 Dose-dependent inhibition of ERK phosphorylation, but not Akt phosphorylation by Bi-Ab in SKOV-3 cells in the absence of IGF-I. Bi-Ab: 200, 40, 8, 1.6, 0.32, and 0 μg/mL.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 10 Inhibition of SKOV-3 cell proliferation in MTT assay.

Percentage of inhibition at each antibody concentration was used in one-way ANOVA statistical analysis to test whether there was significant difference between any two antibodies at the same concentration. Two paired antibodies with significant difference (*, P < 0.001) in percentage of inhibition are indicated.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 11 Percentage of ADCC of the antibodies at 1 and 5 μg/mL.

The same ANOVA statistical analysis was done to test whether there was significant difference in percentage of ADCC between any two antibodies at the same concentration. Percentage of ADCC between Bi-Ab and Comb at 1 μg/mL showed significant difference (*, P < 0.05), which is indicated. Each antibody dilution was tested in triplicate and the assays were repeated once.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 12 Diagram of the mouse study and tumor growth kinetics in each group of mice treated with or without antibodies (control).

A, diagram of the mouse study. Three million of SKOV-3-Luc cells were injected subcutaneously into each nude mouse on day 0. 100 μg of Bi-Ab, or m590, or trastuzumab, or Comb were injected intraperitoneally on day 1, 4, 6, and 8 postinoculations. Mouse imaging was performed on day 1, 4, 6, and 8 before antibody injections, and on day 11, 15, 25, and 35 postinoculations. Seven mice were included in each antibody-treated group, but only five mice were in the control group. B, average luminescence intensity in each group of mice at different time point. Logarithmic values of the average luminescence intensities and standard variations were shown.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 13 Inhibition of cancer growth by Bi-Ab in SKOV-3 HER2- and IGF-IR-overexpressing xenograft mouse model in comparison with m590 and trastuzumab alone, or in combination (Comb).

A, average luminescence intensity in each group of mice at different time point. Logarithmic values of the average luminescence intensity were used in ANOVA statistical analysis to test whether there was significant difference between any two groups at the same time point. Two paired groups with significant difference (*, P < 0.001) are indicated. B, number of mice in each group with luminescence intensity 2-fold higher than the baseline level.

Chen, C., Zhang, Y., Zhang, Y., Li, J., Tsao, S. W., & Zhang, M. Y. (2014). Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor ReceptorBispecific Antibody Cotargeting HER2 and IGF-IR. Molecular cancer therapeutics, 13(1), 90-100.

Figure 14 Binding of mouse IgG2b 4G11 and chimeric IgG1 m590 to non-denatured and denatured IGF-IR and IR ectodomains.

Both ectodomains were coated at 200 ng/well on Maxisorp plates. Denatured ectodomains were prepared as described in Materials and Methods. After blocking the plates with 3% BSA in PBS, three-fold serially diluted antibodies with a starting concentration of 20 µg/ml were added to the plates and bound antibodies were detected with anti-mouse IgG conjugated to HRP for 4G11 and anti-human Fc conjugated to HRP for m590.

Zhang, M. Y., Feng, Y., Wang, Y., & Dimitrov, D. S. (2009, September). Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor. In MAbs (Vol. 1, No. 5, pp. 475-480). Taylor & Francis.

Figure 15 Binding of human-mouse chimeric IgG1 m590, mouse IgG2b 4G11, anti-IGF-IR b subunit antibody GRII and control antibody anti-HIV-1 IgG1 X5 to NWT C43 cells and MCF-7 cells.

Ten 10 mg/ml of each antibody were incubated with stably transfected NWT C43 cells or breast cancer cell line MCF-7. Bound chimeric IgG1 m590 and control antibody IgG1 X5 were revealed by PE labeled anti-human IgG, F(ab')2, mouse IgG2b 4G11 and GRII by PE labeled anti-mouse polyclonal antibody.

Zhang, M. Y., Feng, Y., Wang, Y., & Dimitrov, D. S. (2009, September). Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor. In MAbs (Vol. 1, No. 5, pp. 475-480). Taylor & Francis.

Figure 16 Inhibition of IGF-I and IGF-II induced phosphorylation of IGF-IR by anti-IGF-IR human-mouse chimeric antibody IgG1 m590 and parental murine antibody IgG2b 4G11 in MCF-7 cells.

MCF-7 cells starved in serum-free medium were pre-incubated with indicated concentrations (in nM) of IgG1 m590 (Lanes 3 to 6) and IgG2b 4G11 (Lanes 7 to 9) for 30 min followed by addition of 1.5 nM IGF-I (left) or 10 nM IGF-II (right) for 20 min. Total IGF-IR was immunoprecipitated with rabbit anti-IGF-IR beta subunit pAb. Phosphorylated IGF-IR was detected with mAb 4G10 specific to phosphotyrosine (top gels). The blots were re-probed with rabbit anti-IGF-IR beta subunit pAb (bottom gels) to show the total IGF-IR protein among the samples. Cells treated with IGF-I or IGF-II alone (Lane 2 in both panels) were included as positive control.

Zhang, M. Y., Feng, Y., Wang, Y., & Dimitrov, D. S. (2009, September). Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor. In MAbs (Vol. 1, No. 5, pp. 475-480). Taylor & Francis.