Anti-Human IL6 Recombinant Antibody (Siltuximab) (CAT#: TAB-212)

Figure 1 In vitro effects of siltuximab on ovarian cancer cells 2A.

In vitro inhibition of IL-6 release in IGROV-1 and TOV21G ovarian cancer cells by siltuximab (10-100μg/ml) for three days 2B. Protein was extracted from IGROV1 and TOV21G cells treated with siltuximab (Sil) or IgG control and blotted for expression of Tyr705 phospho-STAT3. 2C. In vitro inhibition of IL-6 release in both IGROV-1 and TOV21G cell lines also led to reduced release of other inflammatory cytokines and chemokines. Typical results of two experiments performed in IGROV1 cells are presented. 2D. Expression of Jagged-1 in unstimulated ovarian cancer cells was assessed by quantitative RT-PCR and immunoblot (left). Following stimulation with either 20 ng/ml IL-6, IgG control or siltuximab for 48 hours, Jagged-1 expression was assessed by quantitative RT-PCR (right). Data are representative of three independent experiments performed. *p<0.05.

Coward, J. , Kulbe, H. , Chakravarty, P. , Leader, D. , Vassileva, V. , & Leinster, D. A. , et al. (2011). Interleukin-6 as a therapeutic target in human ovarian cancer. Clinical Cancer Research, 17(18), 6083-6096.

Figure 2 Actions of siltuximab on intraperitoneal tumors formed from IGROV-1 cells.

Luciferase bioluminescence imaging was used to measure intraperitoneal tumor burden.Siltuximab (20mg/kg twice weekly) treatment for four weeks started 1 day (left) or 14 days(right) after tumor cell injection, and significantly reduced tumor burden compared to IgGcontrol (* p < 0.05). All mice were killed after four weeks of treatment.

Coward, J. , Kulbe, H. , Chakravarty, P. , Leader, D. , Vassileva, V. , & Leinster, D. A. , et al. (2011). Interleukin-6 as a therapeutic target in human ovarian cancer. Clinical Cancer Research, 17(18), 6083-6096.

Figure 3 Actions of siltuximab on intraperitoneal tumors formed from IGROV-1 cells.

Effects of siltuximab on IL-6, phospho-STAT3 and Jagged1 expression in the IGROV-1xenograft model following 4 weeks of siltuximab. The number of tumor cell nuclei showingpositive staining for pSTAT3 were counted in 3 randomly selected areas per tumor section(n=3) using a x40 objective with approximately 500 nuclei counted per tumor (***; p <0.0001). After 4 weeks of siltuximab treatment, there were also marked decreases in bothhuman IL-6 and Jagged1 mRNA expression. RNA from 3 tumor samples in each group wasused for this analysis. In addition, there was a reduction in Jagged-1 expression as detectedby immunohistochemistry. Main photomicrographs taken with x10 magnification lens, insetx40.

Coward, J. , Kulbe, H. , Chakravarty, P. , Leader, D. , Vassileva, V. , & Leinster, D. A. , et al. (2011). Interleukin-6 as a therapeutic target in human ovarian cancer. Clinical Cancer Research, 17(18), 6083-6096.

Figure 4 Phase II trial of the anti-IL-6 antibody siltuximab – survival, clinical responses andpharmacokinetics.

4A. 18 women with recurrent, platinum-resistant ovarian cancer received bi-weeklyinfusions of siltuximab. Patients were re-staged after 3 (12 patients) or 5 (6 patients) dosesand every 12 weeks thereafter. Those achieving Stable Disease (SD) after 3 – 5 dosescontinued treatment for up to 17 infusions. The median progression-free and overallsurvival, PFS and OS respectively, of the patients who received at least one infusion ofsiltuximab was 12 and 49 weeks respectively.4B. CA125 was measured at enrolment and prior to each infusion of siltuximab. Patient 13had a CA125 response by GCIG criteria. PET/CT images at baseline, Week 9 (5 cycles),Week 23 (12 cycles) indicated reduction in [18F]-FDG uptake in pelvic tumors. The regionof high [18F]-FDG uptake anteriorly on the week 23 scan represents the bladder. ULN:Upper Limit of Normal4C. CA125 values prior to and during siltuximab treatment. Patients 5, 9 and 10 hadreductions in CA125 lasting up to 12 weeks. In patient 20, there was a highly significantchange in CA125 doubling time slope after commencing treatment.4D. Siltuximab pharmacokinetics. Serum siltuximab levels were measured immediatelyprior to (Cmin) and one hour after (Cmax) the first three doses of siltuximab.

Coward, J. , Kulbe, H. , Chakravarty, P. , Leader, D. , Vassileva, V. , & Leinster, D. A. , et al. (2011). Interleukin-6 as a therapeutic target in human ovarian cancer. Clinical Cancer Research, 17(18), 6083-6096.

Figure 5 IL-6–dependent upregulation of pStat3 expression and effect of siltuximab.

A, Hamster kidney BHKpEGFP-Stat3 and ovarian cancer cell line SKOV-3 cells were treated with 30 ng/mL IL-6 for 1 hour as indicated. B, Cell lysates were subjected to Western blot analysis with anti- pStat3, or anti-b-actin antibodies as described in Materials and Methods.

Guo, Y. , Nemeth, J. , O"Brien, C. , Susa, M. , Liu, X. , & Zhang, Z. , et al. (2010). Effects of siltuximab on the il-6-induced signaling pathway in ovarian cancer. Clinical Cancer Research, 16(23), 5759-5769.

Figure 6 Confirmation of (A) UGT2B4 and (B) ATP2B2 mRNA expression in IL-6/ siltuximab treated cell lines by real-time RT-PCR.

Real-time RT-PCR was performed as described in Materials and Method. The relative levels of gene expression were calculated from the relevant signals by normalization with the signal for actin expression.

Guo, Y. , Nemeth, J. , O"Brien, C. , Susa, M. , Liu, X. , & Zhang, Z. , et al. (2010). Effects of siltuximab on the il-6-induced signaling pathway in ovarian cancer. Clinical Cancer Research, 16(23), 5759-5769.

Figure 7 Effect of siltuximab on paclitaxel sensitivity in ovarian cancer drug resistant cells.

SKOV-3TR and Caov-3TR cells at 2 Â 103 cells per well were plated in 96-well plates in culture medium containing different concentrations of siltuximab and paclitaxel. After 96 hours of culture, effect of siltuximab on paclitaxel-induced cell death were determined by MTT assay as described in Materials and Methods. A, Effect of siltuximab on paclitaxel sensitivity in SKOV-3TR. C, Effect of siltuximab on paclitaxel sensitivity in Caov-3TR. Statistical analysis was performed by a 2-sided Student's t-test in SKOV-3TR (B) and Caov-3TR (D). Cell viability was assessed using the MTT and recorded as percent viability relative to the untreated control cells. Error bars are SD of averaged results and P < 0.05 were accepted as a significant difference between means. Values are representative of triplicate determinations in 2 or more experiments.

Guo, Y. , Nemeth, J. , O"Brien, C. , Susa, M. , Liu, X. , & Zhang, Z. , et al. (2010). Effects of siltuximab on the il-6-induced signaling pathway in ovarian cancer. Clinical Cancer Research, 16(23), 5759-5769.

Figure 8 Siltuximab inhibits IL-6–induced EGFP-Stat3 nuclear translocation.

SKOV-3 pEGFP-Stat3 and BHKpEGFP-Stat3 cells which stably express the EGFP-Stat3 fusion protein were incubated for 4 hours with siltuximab (0, 0.001, 0.1, 1.0, 10.0 μg/mL) followed immediately thereafter with the addition of IL-6 to a final concentration of 30 ng/mL. Subcellular localization of the fusion protein was assessed by fluorescence microscopy. A, Effects of siltuximab on IL-6–induced stat3 nuclear translocation in BHKpEGFP-Stat3 cells. B, Effects of siltuximab on IL-6–induced stat3 nuclear translocation in SKOV-3pEGFP-Stat3 cells. Arrow labels () show the IL-6–induced Sta3 nucleocytoplasmic shuttling and arrow labels () show the effect of siltuximab (at different doses) on the IL-6–induced Stat3 nuclear translocation.

Guo, Y. , Nemeth, J. , O"Brien, C. , Susa, M. , Liu, X. , & Zhang, Z. , et al. (2010). Effects of siltuximab on the il-6-induced signaling pathway in ovarian cancer. Clinical Cancer Research, 16(23), 5759-5769.

Figure 1 Anti-Human IL6 Recombinant Antibody (TAB-212) in ELISA

ELISA analysis of TAB-212 was performed by coating with human IL6 protein (His tag). Then blocked with BSA and incubated with TAB-212. The HRP-conjugated goat anti-IgG was used as a secondary antibody (1: 2000). Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 2 Anti-Human IL6 Recombinant Antibody (TAB-212) in WB

Western blot analysis of TAB-212 was performed by loading human IL6 protein onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with TAB-212 and a HRP goat anti-IgG (H+L) was used as the secondary antibody (1: 3000). Chemiluminescent detection was performed.

Figure 3 Anti-Human IL6 Recombinant Antibody (TAB-212) in DB

Dot Blot analysis of TAB-212 was performed by coating with human IL6 protein (His tag).
TAB-212 incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-IgG (H+L) secondary antibody (1: 3000)
NC: Negative control, PC: Positive control