Afuco™ Anti-Human FLT3 ADCC Recombinant Antibody (IMC-EB10), ADCC Enhanced (CAT#: AFC-278CL)

Figure 1 IMC-EB10 and IMC-NC7 can inhibit or activate FLT3 phosphorylation along with downstream STAT5, Akt, and MAPK signaling.

A, ALL-derived cell lines were stained with CD135-PE or isotype control and analyzed by FACS. B, ALL-derived cell lines expressing wt FLT3 or FLT3-D835H (on Hb1119 cells) were treated with PBS (0), 10 μg/mL IMC-EB10 (E), or 10 μg/mL IMC-NC7 (N) for 1 hour. Immunoprecipitates and total protein extracts were resolved by 8% or 10% SDS-PAGE, respectively, and subjected to immunoblot analysis with the indicated phospho-specific antibodies. The same blots were then stripped and reprobed with protein-specific antibodies.

Piloto,O.,Nguyen,B.,Huso,D.,Kim,K.T.,Li, Y.,Witte,L.,& Small,D.(2006).IMC-EB10,an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.Cancer research, 66(9), 4843-4851.

Figure 2 IMC-EB10 significantly reduces engraftment of SEM-K2 cells in NOD/SCID mice.

NOD/SCID mice given 0.5 × 10⁶ SEM-K2 cells via tail vein injection were injected i.p.with 400 μg IMC-C225 thrice weekly starting 24 hours after cell injection as control treatment or with 400 μg IMC-EB10 thrice weekly starting 24 hours after cell injection, once starting 24 hours after cell injection, thrice every other day starting 24 hours after cell injection, or thrice weekly starting 7 days after cell injection. Thirty days after cell injection, mice were killed, and the presence of human cells was determined by flow cytometry using human-specific CD19-FITC and CD45-APC antibodies:(A) peripheral blood, (B) bone marrow, and (C) spleen.

Piloto,O.,Nguyen,B.,Huso,D.,Kim,K.T.,Li, Y.,Witte,L.,& Small,D.(2006).IMC-EB10,an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.Cancer research, 66(9), 4843-4851.

Figure 3 IMC-EB10 prolongs survival of NOD/SCID mice given SEM-K2 cells.

NOD/SCID mice given 0.5 × 10⁶ SEM-K2 cells via tail vein injection were injected i.p. with 400 μg IMC-C225 thrice weekly starting 24 hours after cell injection as control treatment or 400 μg IMC-EB10 thrice weekly starting 24 hours after cell injection, once starting 24 hours after cell injection, thrice every other day starting 24 hours after cell injection, or thrice weekly starting 7 days after cell injection. A, mice were monitored daily for survival. B, mouse survival data from all IMC-EB10 treatment regimens combined and compared with IMC-C225 treatment. C, mice surviving 150 days after cell injection were killed and bone marrow and spleen cells analyzed by quantitative real-time PCR to determine the presence of human cells. A standard curve, where murine spleen/bone marrow cells were mixed with SEM-K2 cells at fixed ratios, was used to estimate the percentage of human cells.

Piloto,O.,Nguyen,B.,Huso,D.,Kim,K.T.,Li, Y.,Witte,L.,& Small,D.(2006).IMC-EB10,an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.Cancer research, 66(9), 4843-4851.

Figure 4 IMC-EB10 significantly reduces engraftment of primary ALL blasts in NOD/SCID mice.

NOD/SCID mice received 1 × 10⁶ primary ALL blast via tail vein injection and were treated with 400 μg IMC-EB10 i.p. thrice weekly starting 24 hours after cell injection. Control mice received 200 μL PBS or 400 μg IMC-C225 i.p. thrice weekly starting 24 hours after cell injection. A, 14 weeks after cell injection, human engraftment was determined by flow cytometry using human-specific CD19-FITC and CD45-APC antibodies. B, bone marrow sections of mice were stained with H&E for morphology. C, ALL blasts were treated in vitro with 10 μg/mL IMC-C225 or IMC-EB10 for 1 hour. Immunoprecipitates and total protein extracts were resolved by 8% or 10% SDS-PAGE, respectively, and subjected to immunoblot analysis with the indicated phospho-specific antibodies. The same blots were then stripped and reprobed with protein specific antibodies.

Piloto,O.,Nguyen,B.,Huso,D.,Kim,K.T.,Li, Y.,Witte,L.,& Small,D.(2006).IMC-EB10,an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.Cancer research, 66(9), 4843-4851.

Figure 5 IMC-EB10 treatment of NOD/SCID mice given SEM-K2 cells do not select for resistant clones.

NOD/SCID mice were injected via tail vein with 0.5 × 10⁶ SEM-K2 cells grown in culture without IMC-EB10 or surviving IMC-EB10 treatment within NOD/SCID mice. Mice were injected once i.p. with 400 μg IMC-C225 or IMC-EB10 24 hours after cell injection. Thirty days after cell injection, mice were killed, and their (A) bone marrow and (B) spleen cells analyzed by flow cytometry for the presence of human cells using human-specific CD19-FITC and CD45-APC antibodies.

Piloto,O.,Nguyen,B.,Huso,D.,Kim,K.T.,Li, Y.,Witte,L.,& Small,D.(2006).IMC-EB10,an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.Cancer research, 66(9), 4843-4851.

Figure 6 IMC-EB10 mediates cytotoxicity in vivo through NK cells.

A, NOD/SCID mice were injected with 0.5 × 10⁶ SEM-K2 cells via tail vein and treated with 400 μg IMC-EB10 IP 24 hours after cell injection and/or with 30 μL of Asialo GM1 antisera given at days −3, +4, and +11 relative to when SEM-K2 cells were injected. Control mice were injected with 400 μg IMC-C225 i.p. 24 hours after cell injection. Mice were killed 30 days after cell injection and analyzed for human engraftment using flow cytometry and human-specific CD19-FITC and CD45-APC antibodies. B, NOD/SCID mice were injected with 0.5 × 106 RS411 cells via tail vein and treated with three doses of 400 μg IMC-C225 or IMC-EB10 i.p. every other day starting 24 hours after cell injection. Five days after cell injection, 125 μg poly (I:C) was injected i.p. Forty days after cell injection, mice were killed and analyzed for human engraftment using flow cytometry and human-specific CD19-FITC and CD45-APC antibodies.

Piloto,O.,Nguyen,B.,Huso,D.,Kim,K.T.,Li, Y.,Witte,L.,& Small,D.(2006).IMC-EB10,an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.Cancer research, 66(9), 4843-4851.

Figure 7 Binding and blocking activities of IMC-EB10.

(A) Plates precoated with an anti-His antibody were incubated with His-tagged FLT3-Fc protein for 1 hour. After washing, titrated IMC-EB10 or control antibody IMC-C225 was added and incubated for 1 hour. Bound antibodies were measured in a biotin-streptavidin reaction with a microplate reader at OD450 nm. IMC-EB10 is shown to bind to the FLT3 receptor in a dose-dependent manner. (B) Antibody competition assay of ligand-receptor binding was performed in plates coated with FL. Mixtures of biotinylated FLT3 receptor (fixed amount) and antibody (titrated concentrations) were then added and incubated for 1 hour. FLT3 receptor bound to FL was measured in a biotin-streptavidin reaction with a microplate reader at OD450nm. IMC-EB10 shows a strong blocking activity toward FL-FLT3 receptor binding. Error bars represent means ± SD.

Li,Y.,Li,H.,Wang, M. N.,Lu, D.,Bassi,R.,Wu, Y.,& Kussie,P.(2004).Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody. Blood,104(4), 1137-1144.

Figure 8 IMC-EB10 binds to cell-surface FLT3 receptor in leukemia cells.

Cells were incubated on ice with IMC-EB10 (solid line) or human IgG isotype control (dotted line) and then with PE-conjugated antihuman F(ab′)2 antibody. IMC-EB10 bound to the EOL-1 and BaF3-ITD cell lines, but not to the FLT3-negative cell lines JM1 or BaF3-control. Preincubation of EOL-1 or BaF3-ITD cells for 30 minutes on ice with FL (100 ng/mL) before antibody staining abrogated IMC-EB10 binding to cell surface FLT3 receptor.

Li,Y.,Li,H.,Wang, M. N.,Lu, D.,Bassi,R.,Wu, Y.,& Kussie,P.(2004).Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody. Blood,104(4), 1137-1144.

Figure 9 IMC-EB10 inhibits FL-induced phosphorylation of wild-type FLT3 and ligand-independent constitutive phosphorylation of FLT3-ITD.

(A) EOL-1 and (B) EM3 cells were incubated in varying concentrations of antibody in the presence or absence of FL (30 ng/mL). Lanes 1 and 2 (from right) indicate that a low level of FLT3 phosphorylation existed in untreated EOL-1 cells and that ligand strongly increased the level of receptor phosphorylation. Ligand-induced receptor phosphorylation was blocked by IMC-EB10 antibody but not by a control antibody (control, IMC-C225 at 200 nM). (C) BaF3-ITD and (D) MV4;11 cells were incubated in varying concentrations of antibody without exogenous FL. Ligand-independent phosphorylation was inhibited by IMC-EB10 treatment. Total FLT3 protein was unaffected by antibody treatment.

Li,Y.,Li,H.,Wang, M. N.,Lu, D.,Bassi,R.,Wu, Y.,& Kussie,P.(2004).Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody. Blood,104(4), 1137-1144.

Figure 10 IMC-EB10 inhibits phosphorylation of downstream MAP kinase induced by wild-type FLT3 or FLT3-ITD.

(A) EOL-1 and (B) EM3 cells were incubated with varying amounts of antibody with or without exogenous FL (30 ng/mL). MAPK was phosphorylated after FL stimulation (lanes 1 and 2 from right). Control antibody treatment did not affect MAPK phosphorylation (lane 8 from right). In contrast, treatment with IMC-EB10 (1-200 nM) completely inhibited FL-induced MAPK phosphorylation. (C) BaF3-ITD and (D) MV4;11 cells were incubated in varying concentrations of antibody without exogenous FL. Lane 1 (from right) indicates that MAPK was constitutively phosphorylated. Treatment with control antibody did not affect ligand-independent constitutive MAPK phosphorylation (lane 8 from right). In contrast, constitutive MAPK phosphorylation was inhibited by IMCEB10. Total MAPK protein was unaffected by antibody treatment.

Li,Y.,Li,H.,Wang, M. N.,Lu, D.,Bassi,R.,Wu, Y.,& Kussie,P.(2004).Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody. Blood,104(4), 1137-1144.

Figure 11 Effect of IMC-EB10 on the phosphorylation of AKT and Stat5. Cells were incubated with varying amounts of antibody with or without exogenous FL (30 ng/mL).

(A) In EOL-1 cells, AKT phosphorylation was up-regulated after FL stimulation (lanes 1 and 2 from right). Control antibody treatment did not affect AKT phosphorylation (lane 8 from right). In contrast, IMC-EB10 treatment (1-200 nM) completely inhibited FL-induced AKT phosphorylation. (B) In BaF3-ITD cells, constitutive phosphorylation of AKT was strongly inhibited with IMC-EB10 treatment (1-200 nM). (C) In EOL-1 cells, Stat5 was phosphorylated in the absence of FL stimulation (lane 1 from right), and FL stimulation did not significantly increase the level of phosphorylation of Stat5. Incubation with IMC-EB10 did not have any significant effect on Stat5 phosphorylation in this cell line. (D) In BaF3-ITD cells, constitutive phosphorylation of Stat5 was strongly inhibited with IMC-EB10 treatment (4-200 nM). In both cell lines, total AKT or Stat5 proteins were unaffected by antibody treatment.

Li,Y.,Li,H.,Wang, M. N.,Lu, D.,Bassi,R.,Wu, Y.,& Kussie,P.(2004).Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody. Blood,104(4), 1137-1144.

Figure 12 IMC-EB10 inhibits proliferation of EOL-1 and BaF3-ITD cells.

(A) EOL-1 cells were starved in serum-free medium overnight. Cells were resuspended in AIM-V medium and were incubated for 68 hours with varying concentrations of antibodies (0-100 nM) in the presence or absence of exogenous FL (30 ng/mL). In a background control, cells were incubated with medium alone in the absence of exogenous FL. Cell proliferation was measured by [3H]-thymidine incorporation. For EOL-1 cells, background cycle per minute was deduced from the cycles per minute of all experimental samples. Percentage inhibition of FL-induced cellular proliferation was calculated as follows: [(cpm of untreated sample - cpm of antibody-treated sample)/cpm of untreated sample] × 100%. (B) Proliferation assay with BaF3-ITD cells was performed in RPMI 1640 supplemented with 10% FCS in the absence of exogenous FL for all samples. Error bars represent means ± SD.

Li,Y.,Li,H.,Wang, M. N.,Lu, D.,Bassi,R.,Wu, Y.,& Kussie,P.(2004).Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody. Blood,104(4), 1137-1144.