Anti-Human DR5 Recombinant Antibody (Drozitumab) (CAT#: TAB-180)

Figure 1 Drozitumab (DRO) with anti-Fc antibody treatment induces apoptosis in the sensitive RMS cells.

A, the sensitive Rh18 cells were treated with drozitumab or a 1:1 ratio of drozitumab + anti-Fc antibodies at the indicated doses for 72 hours prior to the viability assay (top). Also, Rh18 cells were treated with various doses of drozitumab + anti-Fc for the indicated time points and analyzed immediately for cell viability (bottom). B, selected sensitive and resistant RMS cell lines and primary SkMDC were treated with drozitumab + anti-Fc or placebo + anti-Fc for 72 hours, followed by cell viability assay (top); or clonogenic assay (bottom). C, both sensitive Rh18 and resistant Rh41 cells were treated with indicated antibodies for 4 hours, fixed, stained with DAPI, and analyzed for fragmented nuclei as a marker for apoptotic cells. D, RMS cells were treated with drozitumab + anti-Fc for the indicated time points and analyzed for the cleavage of apoptotic markers, caspases-3 and -8 and PARP.

Kang, Z., Chen, J. J., Yu, Y., Li, B., Sun, S. Y., Zhang, B., & Cao, L. (2011). Drozitumab, a human antibody to death receptor 5, has potent antitumor activity against rhabdomyosarcoma with the expression of caspase-8 predictive of response. Clinical Cancer Research, 17(10), 3181-3192.

Figure 2 Caspase-8 expression is associated with drozitumab (DRO) sensitivity in RMS cells.

A, immunoblot analysis of RMS cell lines for the expression of proteins relevant to DISC and its effectors. B, both sensitive Rh18 and resistant Rh41 cells were treated with drozitumab + anti-Fc for 10 minutes. Following IP via DR5, both the total cell lysate and the precipitated DISC complex were analyzed for FADD and caspase-8 by immunoblot. C, analysis of the expression of proteins relevant to DR5-DISC and its effectors in a panel of RMS tumor samples. Patients 1 and 3 represent ERMS; patients 4, 5, 6, and 8 represent alveolar RMS; patients 2 and 7are RMS of unknown subtype; SKM is normal skeletal muscle. D, caspase-8 staining (brown) of control RMS xenografts, Rh18 and Rh4, and 2 representative ERMS tumors from an RMS tissue array. The staining results for caspase-8 and DR5 of 18 RMS cases are summarized in the table: 11 were DR5+/casp-8+, 4 were DR5+/casp-8−, 3 were DR5−/casp-8−. Further details can be found in Supplementary Table S1. WI (see Materials and Methods).

Kang, Z., Chen, J. J., Yu, Y., Li, B., Sun, S. Y., Zhang, B., & Cao, L. (2011). Drozitumab, a human antibody to death receptor 5, has potent antitumor activity against rhabdomyosarcoma with the expression of caspase-8 predictive of response. Clinical Cancer Research, 17(10), 3181-3192.

Figure 3 Caspase-8 is both necessary and sufficient for mediating drozitumab (DRO) + anti-Fc-induced apoptosis of RMS cells.

A, sensitive Rh18 cells were treated with drozitumab + anti-Fc in the presence of a caspase-8 specific peptide blocker for 72 hours followed by cell viability measurement. B, sensitive Rh18 cells were treated with drozitumab + anti-Fc for 3 hours, with or without the caspase-8 specific inhibitor. Cell lysates were analyzed by immunoblotting for activated caspase-8 and PARP. C, resistant Rh4 cells were transfected with CASP8 and caspase-defective CASP8 mt (C360S) for 2 days. The cells were then treated with drozitumab + anti-Fc for 3 days and analyzed for cell viability (upper). The cells were also analyzed for caspase-8 and PARP cleavage after 5 hours of treatment with drozitumab + anti-Fc (lower). D, resistant Rh4 cells were transfected with CASP8 and CASP8 mt (C360S) for 2 days. Following drozitumab + anti-Fc treatment for 4 hours, the cells were fixed and analyzed for nuclear fragmentation via DAPI (top). The percentage of apoptotic cells is shown (bottom). Statistical significance (P value) between drozitumab + anti-Fc and CASP8 was determined using 2-way ANOVA.

Kang, Z., Chen, J. J., Yu, Y., Li, B., Sun, S. Y., Zhang, B., & Cao, L. (2011). Drozitumab, a human antibody to death receptor 5, has potent antitumor activity against rhabdomyosarcoma with the expression of caspase-8 predictive of response. Clinical Cancer Research, 17(10), 3181-3192.

Figure 4 Drozitumab (DRO) has potent antitumor activity and a specificity predicted by our in vitro results.

A, both sensitive Rh18 and resistant Rh41 were inoculated intramuscularly into SCID mice. After 4 to 5 weeks, when tumors just became palpable, mice were treated with 10 mg/kg i.p. drozitumab or placebo antibody alone once weekly. The size of the tumors was measured and shown (n = 10). B, mice with sensitive Rh18 tumors were treated with drozitumab or placebo for 4 days. Tumors were resected, and stained with H&E and TUNEL for apoptotic cells. C, Kaplan–Meier survival analysis of the Rh18- or Rh41-tumor bearing mice treated with drozitumab or placebo. The administration of drozitumab for Rh18 was stopped after 10 weeks and the mice were followed for another 18 weeks without treatment until all mice that developed visible tumors died.

Kang, Z., Chen, J. J., Yu, Y., Li, B., Sun, S. Y., Zhang, B., & Cao, L. (2011). Drozitumab, a human antibody to death receptor 5, has potent antitumor activity against rhabdomyosarcoma with the expression of caspase-8 predictive of response. Clinical Cancer Research, 17(10), 3181-3192.

Figure 5 Fluorescence microscopy of FcγR-mediated apoptosis.

The pro-apoptotic signal was observed using a fluorogenic caspase 3/7 detection method from DR5 expressing SK-MES-1 cells (target cells) by co-culturing with Jurkat-Fc RI cells (A), Jurkat cells (No Fc RI) (B), and Jurkat-FcRI cells blocked with Fc RI blocking antibody (C). Target cells were stained in red color and caspase 3/7 expressing cells were in green color. Yellow color denotes the overlay. The scale bar (white) is 100 m. The drozitumab concentration for (A), (B), and (C) is the highest concentration, 200 ng/mL. (D) Quantification of caspase 3/7 positive cells in target cells.

Shim, J., Huang, A., & Miller, A. S. (2017). Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors. Journal of immunological methods, 448, 26-33.

Figure 6 Measuring FcγR-mediated apoptosis using different drozitumab cross-linking methods in solution assay format and binding affinity of drozitumab to FcR using SPR.

The pro-apoptotic signal was observed from DR5 expressing SK-MES-1 cells using a fluorogenic caspase 3/7 detection method. The dose response curves were generated with different drozitumab crosslinking reagents (A) and with different Fc Rs in solution. (B). A 4 parameter model was used to fit the points for the dose response curves. Association constants (KA) of different GST-tagged FcRs to drozitumab were measured using SPR (C). Graphs are of one experiment (A) and representative of at least three independent experiments (B). Error bars indicate the standard deviation of three measurements (C).

Shim, J., Huang, A., & Miller, A. S. (2017). Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors. Journal of immunological methods, 448, 26-33.