Afuco™ Anti-Human HLA-DR βchain ADCC Recombinant Antibody (1D09C3), ADCC Enhanced (CAT#: AFC-346CL)

Figure 1 1D09C3 induces time- and dose-dependent cell death of HLA-DR+ cell lines.

A, expression of HLA-DR by JVM-2, GRANTA-519, and SU-DHL-1 cell lines. B, viable cell counts by flow cytometry following incubation with 1D09C3 (2.5 μg/mL, 24 hours). C, cell survival by MTT assay following incubation with 1D09C3 (2.5 μg/mL, 24 hours). D, time-dependent cell death of JVM-2 cells cultured with 1D09C3 (10 μg/mL). E, time-dependent cell death of GRANTA-519 cells cultured with 1D09C3 (10 μg/mL). F, dose-dependent cell death of JVM-2 cells. G, dose-dependent cell death of GRANTA-519 cells. Cell death data shown in (D-G) were obtained by flow cytometry using the Annexin V/PI double staining. Each cell line was tested on three independent experiments. Columns, mean; bars, SE. The murine anti-HLA-DR 10F12 antibody that fails to induce cell death was used in control cultures. Black columns, Annexin V+/PI− cells. White columns, Annexin V+/PI+ plus Annexin V−/PI+ cells.

Carlo-Stella, C., Di Nicola, M., Turco, M. C., Cleris, L., Lavazza, C., Longoni, P., ... & Nagy, Z. (2006). The Anti–Human Leukocyte Antigen-DR Monoclonal Antibody 1D09C3 Activates the Mitochondrial Cell Death Pathway and Exerts a Potent Antitumor Activity in Lymphoma-Bearing Nonobese Diabetic/Severe Combined Immunodeficient Mice. Cancer Research, 66(3), 1799-1808.

Figure 2 1D09C3 does not induce processing of the caspases or PARP.

A, JVM-2 and GRANTA-519 cells were treated with 10F12 (10 μg/mL) or 1D09C3 (10 μg/mL) for 4 to 24 hours. Cytosolic proteins were then separated by SDS-PAGE and analyzed by immunoblotting with anti-caspase-8, anti-caspase-9, anti-caspase-3, and anti-PARP. No processing of the caspases or PARP was observed. CF, cleaved fragments. As positive control for caspase activation, the KMS-11 cell line exposed to soluble tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) was used. B, JVM-2 and GRANTA-519 cells were treated with 10F12 (10 μg/mL, 4 hours), zVAD-fmk (100 μmol/L), 1D09C3 (10 μg/mL, 4 hours), or 1D09C3 plus zVAD-fmk. Treatment with the caspase inhibitor zVAD-fmk was started 1 hour before adding 1D09C3. Following treatments, cell death was assayed by flow cytometry using the Annexin V/PI double staining.

Carlo-Stella, C., Di Nicola, M., Turco, M. C., Cleris, L., Lavazza, C., Longoni, P., ... & Nagy, Z. (2006). The Anti–Human Leukocyte Antigen-DR Monoclonal Antibody 1D09C3 Activates the Mitochondrial Cell Death Pathway and Exerts a Potent Antitumor Activity in Lymphoma-Bearing Nonobese Diabetic/Severe Combined Immunodeficient Mice. Cancer Research, 66(3), 1799-1808.

Figure 3 1D09C3 induces mitochondrial depolarization and generation of ROS.

JVM-2 (A) and GRANTA-519 (B) cells were plated at 1 × 106/mL and incubated with 10F12 (10 μg/mL) or 1D09C3 (10 μg/mL). At the indicated time points, loss of mitochondrial potential was measured using TMRE staining and flow cytometry. JVM-2 (C) and GRANTA-519 (D) cells were plated at 1 × 106/mL and incubated with 10F12 (10 μg/mL) or 1D09C3 (10 μg/mL). At the indicated time points, generation of ROS was measured using dihydroethidium staining and flow cytometry.

Carlo-Stella, C., Di Nicola, M., Turco, M. C., Cleris, L., Lavazza, C., Longoni, P., ... & Nagy, Z. (2006). The Anti–Human Leukocyte Antigen-DR Monoclonal Antibody 1D09C3 Activates the Mitochondrial Cell Death Pathway and Exerts a Potent Antitumor Activity in Lymphoma-Bearing Nonobese Diabetic/Severe Combined Immunodeficient Mice. Cancer Research, 66(3), 1799-1808.

Figure 4 ROS scavenging inhibits 1D09C3-induced mitochondrial membrane depolarization and cell apoptosis.

GRANTA-519 cells were plated at 1 × 106/mL and treated without or with the ROS scavenger Tiron (10 mmol/L). After 1 hour, the appropriate samples were stimulated with 1D09C3 (4 μg/mL, 30 minutes). A, generation of ROS was measured using dihydroethidium staining. B, mitochondrial membrane depolarization was measured using TMRE staining. C, apoptosis was measured by the Annexin V/PI double staining.

Carlo-Stella, C., Di Nicola, M., Turco, M. C., Cleris, L., Lavazza, C., Longoni, P., ... & Nagy, Z. (2006). The Anti–Human Leukocyte Antigen-DR Monoclonal Antibody 1D09C3 Activates the Mitochondrial Cell Death Pathway and Exerts a Potent Antitumor Activity in Lymphoma-Bearing Nonobese Diabetic/Severe Combined Immunodeficient Mice. Cancer Research, 66(3), 1799-1808.

Figure 5 1D09C3 induces activation and mitochondrial localization of JNK and cytosolic release of AIF.

A, GRANTA-519 cells (1 × 10⁶/mL) were incubated without or with 1D09C3 (10 μg/mL) for the times indicated. Then, cytosolic and mitochondrial extracts were obtained and analyzed with antiphospho-JNK or anti-HSP60 antibodies by Western blot. B, GRANTA-519 cells (1 × 10⁶/mL) were incubated without or with 1D09C3 (10 μg/mL). After treatment, cytosolic extracts were obtained and analyzed by Western blot using an anti-AIF antibody. C, GRANTA-519 cells were incubated for 1 hour without or with L-JNKI1 (1 μmol/L) or L-TAT (1 μmol/L) control peptide. Following exposure to 1D09C3 (10 μg/mL, 1 hour), phospho-JNK levels were analyzed. D, GRANTA-519 cells were incubated for 1 hour without or with L-JNKI1 (1 μmol/L) or L-TAT. Following exposure to 1D09C3 (10 μg/mL, 4 hour), cell death was analyzed.

Carlo-Stella, C., Di Nicola, M., Turco, M. C., Cleris, L., Lavazza, C., Longoni, P., ... & Nagy, Z. (2006). The Anti–Human Leukocyte Antigen-DR Monoclonal Antibody 1D09C3 Activates the Mitochondrial Cell Death Pathway and Exerts a Potent Antitumor Activity in Lymphoma-Bearing Nonobese Diabetic/Severe Combined Immunodeficient Mice. Cancer Research, 66(3), 1799-1808.

Figure 6 Kaplan-Meier estimates of overall survival of mice treated with 1D09C3.

A, NOD/SCID mice were xenografted with JVM-2 cells (1 × 10⁶ per mouse). ○, no treatment; •, PBS; ▪, IgG4 at 3 × 1 mg/mouse (days 4, 7, and 9); □, 1D09C3 at 1 × 0.01 mg/mouse (day 4); ⧫, 1D09C3 at 1 × 0.1 mg/mouse (day 4); ◊, 1D09C3 at 1 × 1 mg/mouse (day 4). B, NOD/SCID mice were xenografted with JVM-2 cells (0.25 × 10⁶ per mouse). ○, PBS; •, 1D09C3 at 1 × 1 mg/mouse (day 4); ▪, 1D09C3 at 3 × 1 mg/mouse (days 4, 7, and 9). C, NOD/SCID mice were xenografted with JVM-2 cells (1 × 106 per mouse). ○, PBS; •, 1D09C3 at 1 × 1 mg/mouse (day 4); ▪, 1D09C3 at 3 × 1 mg/mouse (days 4, 7, and 9). D, NOD/SCID mice were xenografted with GRANTA-519 cells (1 × 10⁶ per mouse). ○, PBS; ▪, 1D09C3 at 3 × 1 mg/mouse (days 1, 4, and 7). E, NOD/SCID mice were xenografted with JVM-2 cells (1 × 10⁶ per mouse). ○, PBS; ▪, 1D09C3 (6 × 1 mg/mouse) at 48-hour intervals starting on day 15. F, NOD/SCID mice were xenografted with GRANTA-519 cells (1 × 10⁶ per mouse). ○, PBS; ▪, 1D09C3 (6 × 1 mg/mouse) at 48-hour intervals starting on day 7. Survival was measured from the day of xenografting. For experiments shown in (A), each treatment group contained 10 mice. For experiments shown in (B-F), each treatment group contained 20 mice.

Carlo-Stella, C., Di Nicola, M., Turco, M. C., Cleris, L., Lavazza, C., Longoni, P., ... & Nagy, Z. (2006). The Anti–Human Leukocyte Antigen-DR Monoclonal Antibody 1D09C3 Activates the Mitochondrial Cell Death Pathway and Exerts a Potent Antitumor Activity in Lymphoma-Bearing Nonobese Diabetic/Severe Combined Immunodeficient Mice. Cancer Research, 66(3), 1799-1808.

Figure 7 The mAb 1D09C3 in a dose response experiments in a Non-Hodgkin's Lymphoma Model (Granta-519).

The mAb 1D09C3 exhibits comparable efficacy within a does range of l mg to 2.5 μg /mouse (50 mg to 125 μg/kg). Efficacy titrates between 2,5 μg (full efficacy) and 25 ng/mouse (no detectable efficacy).

Figure 8 Combination of 1 D09C3 and Rituxan in Non-Hodgkin' s Lymphoma (NHL) Model (Granta-519).

The anti-HLA-DR mAb 1D09C3 shows a clear synergism with the anti-CD20 mAb Rituxan in an NHL model. Single therapies with each antibody show comparable efficacies.

Figure 9 Efficacy in different xenotransplant models.

The 1D09C3 mAb is effective in xenotransplant models of Hodgkin' s lymphoma, non-Hodgkin' s lymphoma, multiple myeloma and hairy cell leukemia.