Blood, Vol. 111, Issue 9, 4813-4816, May 1, 2008

Simultaneously targeting CD45 significantly increases cytotoxicity of the anti-CD33 immunoconjugate, gemtuzumab ozogamicin, against acute myeloid leukemia (AML) cells and improves survival of mice bearing human AML xenografts
Blood Walter et al. 111: 4813

Supplemental materials for: Walter et al

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• Figure S1. Effect of BC8 on drug-induced cytotoxicity in NB4 cells in vitro (JPG, 53.1 KB) -
  NB4 cells were incubated with various concentrations of (A) GO or (B) calicheamicin-₁ for 3 days in the presence or absence of various concentrations of BC8 as indicated before cytotoxicity was assessed with PI staining. Results are shown as mean ± SEM from 3 independent experiments. *p
  
  
  
  
  
• Figure S2. Effect of BC8 on unconjugated hP67.6-induced cytotoxicity in human AML cell lines in vitro (JPG, 44.9 KB) -
  (A) ML-1 and (B) HL-60 cells were incubated with various concentrations of unconjugated hP67.6 for 3 days in the presence or absence of various concentrations of BC8 as indicated before cytotoxicity was assessed with PI staining. Results are shown as mean ± SEM from 3 independent experiments.
  
  
  
  
  
  
• Figure S3. Effect of 31A on drug-induced cytotoxicity in human AML cell lines in vitro (JPG, 87.6 KB) -
  ML-1 and HL-60 cells (upper and lower panel, respectively) were incubated with various concentrations of (A) GO or (B) calicheamicin-₁ for 3 days in the presence or absence of various concentrations of the non-binding murine IgG₁ antibody, 31A, as indicated before cytotoxicity was assessed with PI staining. Results are shown as mean ± SEM from 3 independent experiments.
  
  
  
  
  
  
• Figure S4. Effect of BC8 on CD33 internalization and modulation in NB4 cells in vitro (JPG, 26 KB) -
  (A) CD33 endocytosis. NB4 cells were incubated for 30 minutes with medium containing 2.5 µg/mL of unconjugated, unlabeled hP67.6 in ice-water to prevent internalization during the staining procedure. Cells were then washed in ice-cold PBS to remove unbound antibody, resuspended in antibody-free medium, and incubated at 37°C (in 5% CO₂ and air) for various periods of time. Subsequently, cells were chilled and incubated with biotin-conjugated mouse anti-human IgG₄ monoclonal antibody (5 µg/mL), followed by incubation with streptavidin-PE conjugate (5 µg/mL) to detect remaining hP67.6 on the cell surface. One sample that was kept in ice-water was used to determine the starting level of antibody bound to the cell. (B) CD33 modulation. NB4 cells were incubated overnight in the presence or absence of hP67.6 and/or BC8. Cell surface CD33 was then measured by subsequent staining with hP67.6, a biotin-conjugated mouse anti-human IgG₄ antibody, and a streptavidin-PE conjugate, and expressed as arbitrary fluorescence units (AFU). Results are shown as mean ± SEM from 3-5 independent experiments. *p