Afuco™ Anti-Human CD98 ADCC Recombinant Antibody (IGN523), ADCC Enhanced (CAT#: AFC-139CL)

Figure 1 Antitumor activity of anti-CD98 antibodies in Burkitt's lymphoma model ramos, biophysical properties of 18-2A and IGN523 and comparison of their antitumor efficacy in ramos and dau Burkitt's lymphoma xenografts.

a) of note, in studies 1 and 2, rituximab was used as a positive control and elicited a TGI of 88% (p < 0.001) and 68% (p < 0.001), respectively. (b) biacore analysis of 18-2A (murine IgG2a) and IGN523 (human IgG1) on recombinant human CD98 protein. (c) ramos or (d) dau growth curves in mice receiving 18-2A (15 mg/kg), IGN523 (15 mg/kg) or control antibody (control IgG; 15 mg/kg) once weekly intraperitoneally (i.p.) (n = 8); (c) ***p < 0.001 versus control, (d) *p < 0.05; **p < 0.01 relative to control. Treatment started on day of randomization. Starting tumor volumes were 248 ± 112 and 111 ± 38 mm3 for ramos and dau, respectively. Please note no statistical difference between 18-2A and IGN523 treatment groups in ramos. Arrow = administration of antibody; N.S. = statistically not significant; n.a. = not applicable.

Hayes,G.M.,Chinn,L.,Cantor,J.M.,Cairns,B.,Levashova,Z.,Tran,H.,&Ginsberg,M.H.(2015).Antitumor activity of an anti-CD 98 antibody.International journal of cancer, 137(3), 710-720.

Figure 2 Tumor growth inhibition of IGN523 in different tumor models.

a) HL-60, (b) OCI-AML-3, (c) KG-1, (d) IGN-LNG-12 and (e) IGN-LNG-54 growth curves in either NOD/SCID- (c–e) or CB.17-SCID-mice (a and b) receiving IGN523 (15 mg/kg) or control IgG (15 mg/kg). Carboplatin was administered in IGN-LNG-12 and IGN-LNG-54 at 10 mg/kg i.p. (n = 8). (f) anti-CD98 staining of representative IGN-LNG-54 tumors post-treatment. *p < 0.05; ***p < 0.001 relative to control. Starting tumor volumes were 134 ± 32 mm3 (HL-60); 174 ± 34 mm3 (OCI-AML-3); 92 ± 11 mm3 (KG-1); 126 ± 23 mm3 (IGN-LNG-12) and 136 ± 38 mm3 (IGN-LNG-54). Please note a statistical difference between IGN523 and carboplatin treatment groups in IGN-LNG-54 (p < 0.04). Arrow = administration of antibody. Scale bar = 20 μm.

Hayes,G.M.,Chinn,L.,Cantor,J.M.,Cairns,B.,Levashova,Z.,Tran,H.,&Ginsberg,M.H.(2015).Antitumor activity of an anti-CD 98 antibody.International journal of cancer, 137(3), 710-720.

Figure 3 IGN523 demonstrates strong ADCC-,but not CDC-activity.

Antibody-induced luciferase activity in the effector cell is quantified with luminescence readout and is a surrogate for effector cell-mediated cell lysis. Target cells (a and d) ramos, (b and e) KG-1 and (c) OCI-AML-3 were used in (a–c) ADCC or (d and e) CDC assays. Please note that IGN523 elicits greater ADCC activity than rituximab in ramos cells. Cetuximab (anti-EGFR) was used as negative control antibody (control IgG). Ramos, KG-1 and OCI-AML-3 cell lines both endogenously express CD98 on the cell surface.

Hayes,G.M.,Chinn,L.,Cantor,J.M.,Cairns,B.,Levashova,Z.,Tran,H.,&Ginsberg,M.H.(2015).Antitumor activity of an anti-CD 98 antibody.International journal of cancer, 137(3), 710-720.

Figure 4 IGN523 decreases viability, amino acid transport and increases caspase-3 and caspase-7 activity.

Cells were incubated with IGN523 in the presence or absence of AbXL. (a–c) cell viability was assessed by measurement of live-cell protease activity that is restricted to intact viable cells (fluorescence). (d–f) Caspase-3 and caspase-7 activity was measured by increase in aminoluciferin-induced luminescence through DEVD-aminoluciferin substrate cleavage. (g–i) crosslinked IGN523 reduced 3Phe-uptake. BCH was used at 10 mM. The ratio of IGN523:AbXL was 1:5. The amount of 3Phe-uptake in ramos cells under each treatment determined as counts per minute (cpm) is graphed. *p < 0.05; ***p < 0.001 relative to IGN523 (a and b) or control (c), respectively.

Hayes,G.M.,Chinn,L.,Cantor,J.M.,Cairns,B.,Levashova,Z.,Tran,H.,&Ginsberg,M.H.(2015).Antitumor activity of an anti-CD 98 antibody.International journal of cancer, 137(3), 710-720.

Figure 5 IGN523-CD98 complex colocalizes with LAMP-1.

IGN523 induces lysosomal permeability. (a) ramos cells were incubated at 4 °C for 30 min with 2 μg/mL Alexa488-labeled anti-human CD98 antibody (green), 10 μg/mL anti-human crosslinker (AbXL) and hoechst (blue). Cells were subsequently incubated at 37 °C for the indicated time points (0 or 90 min). LAMP1 was stained with anti-LAMP1-AF647 antibody (red). Note that CD98 and LAMP-1 colocalize (yellow). Top row: 0 min; bottom row: 90 min; scale bar = 10 μm. (b) lysosomal volume and (c) lysosomal permeability was assessed in IGN523-crosslinked ramos cells at 4 and 12 hr, respectively. The ratio of IGN523 (10 μg/mL):AbXL (50μg/mL) was 1:5. Cetuximab was used as control antibody (control IgG). ***p < 0.001 relative to IGN523.

Hayes,G.M.,Chinn,L.,Cantor,J.M.,Cairns,B.,Levashova,Z.,Tran,H.,&Ginsberg,M.H.(2015).Antitumor activity of an anti-CD 98 antibody.International journal of cancer, 137(3), 710-720.