NeuroMab™ Anti-CXCR4 BBB Shuttle Antibody(NRZP-1022-ZP3812)

Figure 1 shows EasyCyte staining demonstrating binding of C-9P21, 9N10, and F7 IgG clones to Ramos and CCRF-CEM cell lines.

Figure 2 shows EasyCyte staining of 105 CCRF-CEM cells in which 0.1 μg each of C-9P21, 9N10, and F7 IgG clones did not compete with 4 μg, 1 μg, and 0.5 μg of ligand.

Figure 3 shows the results of a Ca++-flux assay performed on CCRF-CEM cells (labeled with Fluo-4).

Antibody clone 9N10 was tested at the IgG level. Cells were pre-incubated with 4 μg/ml scF vs 10 μg/ml IgG (Figure 11E) for 15 minutes before adding the CXCR4-specific ligand SDF-1α. Signal recording was started approximately 10 seconds after ligand addition. Area under the curve (AUC) was integrated and plotted as a percentage of the AUC of maximal stimulation with SDF-1a alone.

Figure 4 shows the results of a Ca++-flux assay performed on CCRF-CEM cells (labeled with Fluo-4).

Figure 5 shows the inhibition of ligand (SDF-la) induced cell migration by anti-CXCR4 scFv C-9P21.

Human T-cell leukemia CCRF-CEM cells labeled with BATDA were induced to migrate in a Boyden chamber in which SDF-1 ligand was placed in the lower chamber and cells were co-incubated with antibodies or culture medium, serving only as a control chamber for the upper chamber. Cells migrating to the lower chamber were detected by fluorescence intensity after Triton X-100-induced cell lysis. Mean and SD values of triplicates are plotted.

Figure 6 shows the induction of ADCC by anti-CXCR4 antibodies B-1M22, C-9P21, C-1124 and D-1K21.

Dose-response killing of CCRF-CEM cells is shown for scFvs C-9P21 and D-1K21 (a), IgG C-9P21 and D-1K21 (b), and IgG B-1M22 and C-1I24.

Figure 7 shows the results of assays testing the ability of antibodies B-1M22, C-9P21, C-1I24 and D-1K21 to induce CDC.

C-9P21 and D-1K21 showed dose-responsive killing of Ramos cells in the presence of human serum without induction of CDC. As a control, antibody F7 in IgG1 format was used.

Figure 8 shows the analysis of apoptotic activity of anti-CXCR4 antibodies C-9P21 and 9N10 with Annexin V. Antibody F7 was used as a control. Light gray shading total amount of dead cells, positive for both Annexin V and PI, dark gray shading apoptotic cells defined as Annexin V positive but PI negative.