Anti-Human CD80 Recombinant Antibody (Galiximab) (CAT#: TAB-215)

Figure 1 Galiximab inhibits cell proliferation of B-NHL cell lines and sensitizes B-NHL cells to apoptosis by CDDP and TRAIL.

A, surface expression of CD80 on B-NHL cell lines. The surface expression of CD80 was analyzed by flow cytometry. A, representative histogram is shown for the various B-NHL cell lines and an isotype control is shown. In addition, the mean fluorescence intensity (MFI) is represented in the table. B, sensitization to apoptosis. B-NHL cell lines were treated with galiximab (20 mg/mL) for 18 hours and followed by treatment with CDDP (5 mg/mL) or TRAIL (5 ng/mL) for an additional 18 hours and apoptosis was determined by activation of caspase-3 (left) as described. Ã, P < 0.05; ÃÃ, P < 0.01. Apoptosis was also determined by Annexin-V as described (right). C, galiximab-induced inhibition of Raji cell viability and cell recovery. The B-NHL cell line Raji was treated with various concentrations of galiximab and incubated for different time periods (6-24 hours), and cell viability was determined by trypan blue dye exclusion and total cell recovery was recorded. Raji cells that were not treated with galiximab represented 100% viability. The data represent the mean AE SD from 3 independent experiments Ã, P < 0.05. D, galiximab sensitizes resistant B-NHL cell lines to apoptosis by CDDP and TRAIL. Sensitization of B-NHL Raji cells by galiximab to apoptosis by CDDP or TRAIL is synergistic. Raji cells were treated with various concentrations of galiximab (10-100 mg/mL) for 18 hours and then treated with either CDDP (5, 10, and 20 mg/mL; left) or only one concentration of galiximab (20 mg/mL) and various concentrations of TRAIL (2.5, 5, and 10 ng/mL; right) for an additional 18 hours and apoptosis was determined. The data represent the mean AE SD from 3 independent experiments. Ã, P < 0.05; ÃÃ, P < 0.01. In addition, the data were analyzed for synergy by isobologram analysis as described in Materials and Methods. The isobologram is represented left of D. AAD, aminoactinomycin D.

Martinez-Paniagua, M. A., Vega, M. I., Huerta-Yepez, S., Baritaki, S., Vega, G. G., Hariharan, K., & Bonavida, B. (2012). Galiximab Signals B-NHL Cells and Inhibits the Activities of NF-κB–Induced YY1-and Snail-Resistant Factors: Mechanism of Sensitization to Apoptosis by Chemoimmunotherapeutic Drugs. Molecular cancer therapeutics, 11(3), 572-581.

Figure 2 Galiximab inhibits NF-kB activity in Raji cells and the role of NF-kB inhibition in the sensitization of Raji to apoptosis by CDDP and TRAIL.

Raji cells were treated with different concentrations of galiximab (25, 50, and 100 mg/mL) for 18 hours, and aliquots were used to prepare both nuclear and total cell lysates as described in Materials and Methods. A, Western blot analysis for NF-kB expression. Total cell lysates were tested for various gene products of the NF-kB pathway. b-Actin was used as a loading control. Densitometric analysis is also shown and intensity of the bands was normalized to b-actin bands. B, inhibition of NF-kB DNA-binding activity by galiximab. Raji cells were treated with galiximab (25 mg/mL). Nuclear lysates were tested for NF-kB DNAbinding activity by EMSA as described. The NF-kB inhibitor DHMEQ (10 mg/mL) was used as a positive control and cold probes as competitors. For the supershift assay, the nuclear proteins were incubated with anti-p65 antibody overnight at 4 C before the analysis by EMSA. C, galiximab-induced inhibition of NF-kB in the sensitization to apoptosis by CDDP and TRAIL. Raji cells were treated with galiximab (20 mg/mL) for 18 hours or with the NF-kB inhibitor DHMEQ (10 mg/mL) for 18 hours and the cells were subsequently treated with either CDDP (5 mg/mL) or TRAIL (5 mg/mL) for an additional 24 hours and apoptosis was determined. The data represent the mean AE SD from 3 independent experiments. Ã, P < 0.01. D, inhibition of the AKT pathway by galiximab. Total cell lysates were tested for various gene products of the AKT pathway. b-Actin was used as a loading control. Densitometric analysis is also shown.

Martinez-Paniagua, M. A., Vega, M. I., Huerta-Yepez, S., Baritaki, S., Vega, G. G., Hariharan, K., & Bonavida, B. (2012). Galiximab Signals B-NHL Cells and Inhibits the Activities of NF-κB–Induced YY1-and Snail-Resistant Factors: Mechanism of Sensitization to Apoptosis by Chemoimmunotherapeutic Drugs. Molecular cancer therapeutics, 11(3), 572-581.

Figure 3 Galiximab inhibits the expression and the activity of the transcription factors YY1 and Snail in Raji cells.

Inhibition of YY1 and Snail by galiximab. Raji cells were treated with various concentrations of galiximab (25, 50, and 100 mg/mL) for 18 hours and total cell lysates were prepared for Western blot analysis. b-Actin was used as a loading control. The Western blots analyses were also analyzed by densitometry and is shown below the Western blot analysis figure.

Martinez-Paniagua, M. A., Vega, M. I., Huerta-Yepez, S., Baritaki, S., Vega, G. G., Hariharan, K., & Bonavida, B. (2012). Galiximab Signals B-NHL Cells and Inhibits the Activities of NF-κB–Induced YY1-and Snail-Resistant Factors: Mechanism of Sensitization to Apoptosis by Chemoimmunotherapeutic Drugs. Molecular cancer therapeutics, 11(3), 572-581.

Figure 4 Galiximab inhibits cell proliferation of B-NHL cell lines and sensitizes B-NHL cells to apoptosis by CDDP and TRAIL.

A, surface expression of CD80 on B-NHL cell lines. The surface expression of CD80 was analyzed by flow cytometry. A, representative histogram is shown for the various B-NHL cell lines and an isotype control is shown. In addition, the mean fluorescence intensity (MFI) is represented in the table. B, sensitization to apoptosis. B-NHL cell lines were treated with galiximab (20 μg/mL) for 18 hours and followed by treatment with CDDP (5 μg/mL) or TRAIL (5 ng/mL) for an additional 18 hours and apoptosis was determined by activation of caspase-3 (left) as described. *, P < 0.05; **, P < 0.01. Apoptosis was also determined by Annexin-V as described (right). C, galiximab-induced inhibition of Raji cell viability and cell recovery. The B-NHL cell line Raji was treated with various concentrations of galiximab and incubated for different time periods (6–24 hours), and cell viability was determined by trypan blue dye exclusion and total cell recovery was recorded. Raji cells that were not treated with galiximab represented 100% viability. The data represent the mean ± SD from 3 independent experiments *, P < 0.05. D, galiximab sensitizes resistant B-NHL cell lines to apoptosis by CDDP and TRAIL. Sensitization of B-NHL Raji cells by galiximab to apoptosis by CDDP or TRAIL is synergistic. Raji cells were treated with various concentrations of galiximab (10–100 μg/mL) for 18 hours and then treated with either CDDP (5, 10, and 20 μg/mL; left) or only one concentration of galiximab (20 μg/mL) and various concentrations of TRAIL (2.5, 5, and 10 ng/mL; right) for an additional 18 hours and apoptosis was determined. The data represent the mean ± SD from 3 independent experiments. *, P < 0.05; **, P < 0.01. In addition, the data were analyzed for synergy by isobologram analysis as described in Materials and Methods. The isobologram is represented left of D. AAD, aminoactinomycin D.

Martinez-Paniagua, M. A., Vega, M. I., Huerta-Yepez, S., Baritaki, S., Vega, G. G., Hariharan, K., & Bonavida, B. (2012). Galiximab Signals B-NHL Cells and Inhibits the Activities of NF-κB–Induced YY1-and Snail-Resistant Factors: Mechanism of Sensitization to Apoptosis by Chemoimmunotherapeutic Drugs. Molecular cancer therapeutics, 11(3), 572-581.

Figure 5 Galiximab inhibits the expression and the activity of the transcription factors YY1 and Snail in Raji cells.

A, inhibition of YY1 and Snail by galiximab. Raji cells were treated with various concentrations of galiximab (25, 50, and 100 μg/mL) for 18 hours and total cell lysates were prepared for Western blot analysis. β-Actin was used as a loading control. The Western blots analyses were also analyzed by densitometry and is shown below the Western blot analysis figure. B, galiximab inhibits the DNA-binding activity of YY1 and Snail. Raji cells were treated with galiximab (25 μg/mL) for 18 hours and nuclear lysates were tested for DNA-binding activities for YY1 and Snail as described in Materials and Methods. The specificity of DNA-binding activity was determined by the use of a corresponding competitive cold probe and in the absence of nuclear extracts in the assay.

Martinez-Paniagua, M. A., Vega, M. I., Huerta-Yepez, S., Baritaki, S., Vega, G. G., Hariharan, K., & Bonavida, B. (2012). Galiximab Signals B-NHL Cells and Inhibits the Activities of NF-κB–Induced YY1-and Snail-Resistant Factors: Mechanism of Sensitization to Apoptosis by Chemoimmunotherapeutic Drugs. Molecular cancer therapeutics, 11(3), 572-581.