Anti-Human CD40 Recombinant Antibody (Dacetuzumab) (CAT#: TAB-152)

Figure 1 Agonistic activity of SGN-40 on normal B cells.

A, B cells enriched from PBMCs were treated with increasing concentrations of SGN-40 or a nonbinding control IgG (cIgG) with (bottom ) or without (top ) secondary cross-linking (XL) by the F(ab') 2 fragments of a goat antibody specific for the Fcg fragment of human IgG. IL-4 (10 ng/mL) was also tested. Cells were stimulated for a total of 72 hours and proliferation was detected by pulsing with [ 3 H]thymidine during the last 16 hours of incubation. Bars, SDs from triplicate determinations. B, B cells enriched from PBMCs were treated with SGN-40 or a control nonbinding IgG (cIgG) with or with secondary cross-linking. The effects of IL-4 (10 ng/mL), recombinant CD40L (1 Ag/mL), or IL-4 plus recombinant CD40L in combination with SGN-40 were also tested. Cells were stimulated and proliferation as in (A). Bars, SDs from triplicate determinations.

Law, C. L., Gordon, K. A., Collier, J., Klussman, K., McEarchern, J. A., Cerveny, C. G.,... & Wahl, A. F. (2005). Preclinical antilymphoma activity of a humanized anti-CD40 monoclonal antibody, SGN-40. Cancer research, 65(18), 8331-8338.

Figure 2 Binding characteristics of SGN-40 and S2C6.

A, binding of SGN-40 and S2C6 to Ramos B-lymphoma cells was determined by indirect immunofluorescence staining and flow cytometry. Background-subtracted mean fluorescence intensity (DMFI ) was plotted against the mAb concentrations. B, an equilibrium saturation binding assay as described in Materials and Methods was used to determine the K d values for SGN-40 and S2C6 to CD40 expressed by Ramos cells. Representative binding curves generated from the means of three independent experiments are shown. In each experiment, quadruplicate determinations were done for each concentration of labeled antibody used. Points, mean; bars, SE.

Law, C. L., Gordon, K. A., Collier, J., Klussman, K., McEarchern, J. A., Cerveny, C. G.,... & Wahl, A. F. (2005). Preclinical antilymphoma activity of a humanized anti-CD40 monoclonal antibody, SGN-40. Cancer research, 65(18), 8331-8338.

Figure 3 Cytotoxic effects of SGN-40 in CD40-expressing multiple myeloma (MM) cell lines and patient MM cells.

A, Dex-sensitive MM.1S and Dex-resistant MM.1R cell lines (left, □, MM.1S; ▪, MM.1R) and two patient MM cells (right, □, MM#1; ▪, MM#2) were incubated with SGN-40 at 0–100 μg/ml. After 36 h, cells were pulsed with [3H]thymidine for 8 h (MM.1S and MM.1R cell lines) or overnight (patient MM cells), and DNA synthesis was measured. B, MM.1S (squares) and MM.1R (circles) were plated on 96-well plates in triplicate and treated with SGN-40 (0–50 μg/ml, left) or isotype control immunoglobulin (0–50 μg/ml, right) in the presence (solid symbols) or absence (open symbols) of protein synthesis inhibitor cycloheximide (CHX; 0.2 μg/ml). Cell viability was assessed by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay. C, cell viability of two patient MM cells (circles, MM#1; squares, MM#2) treated with SGN-40 (0–50 μg/ml) in the presence (solid symbols) or absence (open symbols) of CHX (0.2 μg/ml). D, RNA was isolated from MM.1S cells treated with SGN-40 for 0 h, 3 h, or 24 h (Lanes 1–3, respectively) and subjected to reverse transcription-PCR analysis for the expression of FasL, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; Apo-2L), and tumor necrosis factor α (TNF-α). GADPH serves as an internal control.

Tai, Y. T., Catley, L. P., Mitsiades, C. S., Burger, R., Podar, K., Shringpaure, R.,... & Richardson, P. (2004). Mechanisms by which SGN-40, a humanized anti-CD40 antibody, induces cytotoxicity in human multiple myeloma cells: clinical implications. Cancer Research, 64(8), 2846-2852.

Figure 4 SGN-40 activates neither AKT nor nuclear factor κB in MM.1S cells.

A, serum-starved MM.1S cells were incubated with SGN-40 (5 μg/ml) or sCD40L (5 μg/ml) for the indicated time intervals. Samples were collected and evaluated by immunoblot analysis with antiphosphorylation-specific antibodies. Detection of total AKT on the same blot was used to determine equal loading of samples. B, serum-starved MM.1S cells were stimulated with SGN-40 at increasing concentrations (0–1000 μg/ml). Samples were collected and evaluated by immunoblot analysis with antiphosphorylation-specific antibodies. α-Tubulin was used as a loading control.

Tai, Y. T., Catley, L. P., Mitsiades, C. S., Burger, R., Podar, K., Shringpaure, R.,... & Richardson, P. (2004). Mechanisms by which SGN-40, a humanized anti-CD40 antibody, induces cytotoxicity in human multiple myeloma cells: clinical implications. Cancer Research, 64(8), 2846-2852.

Figure 5 Nuclear factor κB/AKT activation induced by sCD40L is not altered by SGN-40.

A, serum-starved MM.1S cells were incubated with SGN-40 (5–10 μg/ml) for 7 min in the presence or absence of sCD40L (5–10 μg/ml). Isotype control immunoglobulin (5 μg/ml) was used as a control. Cell lysates were subjected to immunoblot analysis using anti-pAKT, pIκBα, and pERK1/2 antibodies, and with anti-AKT antibody as a loading control. B, serum-starved MM.1S cells were pretreated with various concentrations (5–1000 μg/ml) of SGN-40 for 30 min before stimulation with 5 μg/ml sCD40L (+) for 7 min. Cellular proteins then underwent immunoblot analysis with anti-pAKT and with anti-ΑΚΤ as a loading control.

Tai, Y. T., Catley, L. P., Mitsiades, C. S., Burger, R., Podar, K., Shringpaure, R.,... & Richardson, P. (2004). Mechanisms by which SGN-40, a humanized anti-CD40 antibody, induces cytotoxicity in human multiple myeloma cells: clinical implications. Cancer Research, 64(8), 2846-2852.

Figure 6 Interleukin 6 receptor (IL-6R; gp80) expression in multiple myeloma (MM) cells treated with SGN-40.

A, MM.1S cells (top) and patient MM cells (MM#1; bottom) were treated with 20 μg/ml of SGN40 for 0–9 h; expression of IL-6R then was evaluated by immunoblot analysis in MM.1S cells and by immunoprecipitation followed by immunoblot analysis in MM#1 cells. B, flow cytometric analysis (open histogram) of surface IL-6R expression is shown on MM.1S and patient MM cells (MM#1) treated with 20 μg/ml of either SGN40 or control immunoglobulin overnight. Shaded histogram is isotype control immunoglobulin for anti-IL-6R antibody. C, MM.1S cells were pretreated for 1.5 h with U1026 (1 μm), PD98059 (10 μm), LY294002 (5 μm), and SB203580 (5 μm) before overnight treatment with SGN-40. IL-6R was assayed by immunoblot analysis.

Tai, Y. T., Catley, L. P., Mitsiades, C. S., Burger, R., Podar, K., Shringpaure, R.,... & Richardson, P. (2004). Mechanisms by which SGN-40, a humanized anti-CD40 antibody, induces cytotoxicity in human multiple myeloma cells: clinical implications. Cancer Research, 64(8), 2846-2852.

Figure 7 SGN-40 mediates antibody-dependent cellular phagocytosis (ADCP) activity and requires functional Fc–FcγR interaction in vitro.

(A) Binding of SGN-40 and SGN-40G1v1 to CD40+ Ramos cells was detected by flow cytometry. EC50 values are 0.510 and 0.528 nM for SGN-40 and SGN-40G1v1, respectively. (B) Binding of SGN-40 and SGN-40G1v1 to CHO DG-44 cell lines expressing human or mouse FcγRI (CD64) and FcγRIIIA V158 (CD16) as determined by flow cytometry. (C) SGN-40 induces ADCP activity in vitro as determined by flow cytometry and fluorescence microscopy. (D) CD40-positive Ramos, WIL2-S and the CD40-negative L540cy target cells were labelled with PKH26 lipophilic dye, treated with varying concentrations of SGN-40, SGN-40G1v1 or non-binding control IgG then mixed with Mø. (E) Survival curve of mice implanted with Ramos tumour cells and left untreated or following treatment with 4 mg kg−1 SGN-40 or SGN-40G1v1 on day 1 (n=10 per group), untreated vs SGN-40 (P<0.0001), untreated vs SGN-40G1v1 (P=0.7190), SGN-40 vs SGN-40G1v1 (P<0.0001). Data shown are from one representative of a total of two independent experiments.

Oflazoglu, E., Stone, I. J., Brown, L., Gordon, K. A., Van Rooijen, N., Jonas, M.,... & Gerber, H. P. (2009). Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40. British journal of cancer, 100(1), 113.

Figure 8 Macrophages mediate antitumour activity of SGN-40 and requirement for intact Fc–FcγR interactions in vivo.

(A) Mice implanted with Ramos tumours (NHL) were depleted of natural killer cells (−NK) (B) neutrophils (−Neut), (C) macrophages (−Mac) or (D) all subsets combined (−All). Mice were either left untreated or treated with 4 mg kg−1 SGN-40 on day 1 post tumour implantation (n=10 per group), using intraperitoneal injections. Untreated vs SGN-40 (P<0.0001), untreated vs −NK+SGN-40 (P<0.0001), SGN-40 vs −NK+SGN-40 (P=0.4611). Untreated vs −Neut+SGN-40 (P=0.0001), SGN-40 vs −Neut+SGN-40 (P=0.5969). Untreated vs −Mac+SGN-40 (P=0.002), SGN-40 vs −Mac+SGN-40 (P=0.0016). Untreated vs All depleted (P=0.0594), SGN-40 vs All depleted +SGN-40 (P=0.0004). Data shown are from one representative of a total of two or three independent experiments. (E) FACS analysis of spleen from NK cell-depleted mice using anti-DX5 antibody, or peripheral blood from neutrophil-depleted mice, using an anti-CD11b antibody. The right panels display IHC analysis of liver tissues isolated from macrophage-depleted mice using an anti-mouse-F4/80 antibody.

Oflazoglu, E., Stone, I. J., Brown, L., Gordon, K. A., Van Rooijen, N., Jonas, M.,... & Gerber, H. P. (2009). Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40. British journal of cancer, 100(1), 113.

Figure 1 Anti-CD40 Antibody (TAB-152) in WB

Western blot analysis of TAB-152 was performed with human CD40 protein.

Figure 2 Anti-CD40 Antibody (TAB-152) in ELISA

ELISA analysis of TAB-152 was performed by coating with human CD40 protein.

Figure 3 Anti-CD40 Antibody (TAB-152) in Dot Blot

Dot Blot analysis of TAB-152 was performed by coating with human CD40 protein.