NeuroMab™ Anti-CXCL13 BBB Shuttle Antibody(NRZP-1022-ZP3438)

Figure 1. Epitope competition ELISA results showing percent inhibition of binding of biotinylated 3D2 to human CXCL13 by mouse anti-human CXCL13 antibodies (3C9 and 3D2) compared to results without competitor or MAb 801.

Figure 2. Capture epitope competition ELISA results show that 3D2 inhibits the binding of biotin 3C9 to native or recombinant human CXCL13 (A) and the binding of biotin 3D2 to native or recombinant mouse CXCL13 (B).

Curves were fitted using four-parameter sigmoidal curve fit (curves are shown on the graph; the R2=0.99). The differences in EC50 values were analyzed by unpaired t-test and were found to be P>0.05.

Figure 3. B cell migration results show the effects of 3D2 and 3C9 on human CXCL13-induced migration of human pre-B-697-hCXCR5 cells (A) and human SDF-1α-induced migration of human pre-B-697-hCXCR4 cells (B).

Mouse IgG was used as a negative control. MAb 801 was used as a positive control for inhibition of human CXCL13 migration, and MAb 87A was used as a positive control for inhibition of human SDF-1 alpha-induced migration.

Figure 4. Effects of 3D2, MAb 470, mouse IgG (control) or rat IgG (control) in C57Black/6 (A) and 3D2, 3C9, MAb 470 or mouse IgG (control) in SJL/J on splenocyte migration Inhibition percentage) (B). Results are expressed as the mean of two (C57Black/6 migration) independent experiments +/- SD and three (SJL/J migration) independent experiments +/- SEM. The comparison of the effect of 3D2 on C57Black/6 and SJL/J migration (C) was analyzed by unpaired t-test, which yielded P values >0.05. Curves were fitted using a four-parameter sigmoidal curve fit (curves are shown on the graph; R2 = 0.99).

Figure 5. Human CXCL13-mediated endocytosis of human and mouse CXCR5 receptors (A) and mouse CXCL13-mediated endocytosis (B) of CXCL13 by 3D2 or control (MAb 470 and/or mouse IgG) mediated endocytosis. Comparative EC50 values for human and mouse CXCL13-mediated endocytosis were calculated from sigmoidal dose-response curves with R2 values equal to 1 (mouse endocytosis) and 0.994 (human endocytosis), as shown (C). Data comparing the effects of 3D2 on human and mouse receptor endocytosis were analyzed by unpaired t-tests yielding P values >0.05.

Figure 6. EAE disease progression in mice treated with 3D2 (from day 0), 3D2 (from score >1), or mouse IgG control (RR-EAE-1 study). Each data point represents the average score of 9 mice. Group means (GMS) were compared by using one-way ANOVA followed by Bonferroni's post-test for multiple comparisons.

Figure 7. EAE Disease in Mice Treated with 3D2 (From Day 0), 3D2 (From Day 6), 3D2 (From Day >2), or Mouse IgG Control (RR-EAE-2 Study) progress. Each data point represents the average score of 9 mice. Group means (GMS) were compared by using one-way ANOVA followed by Bonferroni's post-test for multiple comparisons.

Figure 8. Renal pathology in mice with advanced lupus nephritis after 3D2 or mouse IgG (control) treatment (SLE-1 study). For proteinuria scores (A) and renal pathology scores (B) for glomerulonephritis, interstitial nephritis, and vasculitis, each data point represents the mean of ten measurements.

Figure 9. Renal pathology in mice with early lupus disease after 3D2 and mouse IgG (control) treatment (SLE-2 study). For proteinuria scores (A) and renal pathology scores for glomerulonephritis and interstitial nephritis (B), each data point represents 7 mice from the 3D2-treated group and 9 mice from the mouse IgG-treated group mice.

Figure 10: Histological sections showing the effect of 3D2 on the number of germinal centers (GC) and primary follicles in the spleen of lupus mice. Spleen sections stained with GL-7 (GC stain), B220 antibody (B cell marker), or follicular dendritic cells (FDC) from 3D2-treated (A) and mouse IgG-treated (B) NZB/NZWF1 mice Antibody staining.

Figure 11. Primary follicles and GC size in the spleen of lupus mice treated with 3D2. Values are shown as mean +/- SEM of 5 mice per group. Mice treated with 3D2 ("tx") showed a trend toward reduced number of GCs (p=0.19) (A) and a significant reduction in GC size (p =0.03) (2).

Figure 12. Epitope competition ELISA results of MAb 5261, MAb 5080 and 3D2 binding to native human (A) and native mouse (B) CXCL13.

Each data point represents an average of duplicate measurements from one of at least three independent experiments. Curves were fitted using four-parameter sigmoidal curve fit (curves are shown on the graph; R2=0.99).

Figure 13. Specific ELISA results for binding of MAb 5378, 3D2, and mouse isotype control to recombinant human (A), cynomolgus monkey (B) and mouse (C) CXCL13.

Each data point represents the mean of triplicate measurements. EC50 values were calculated from a four-parameter sigmoidal curve fit (curves shown; R2 of the curve yielding EC50 values was 0.99).

Figure 14. Percent Inhibition of Migration of Human Pre-B-697-hCXCR5 (24A), Human Tonsil Cells (24B) and C57Black6 Mouse Splenocytes (24C) by MAb 5261 or MAb 5378 (24A-B) and MAb 5378 or 3D2 (24C) . Data represent mean +/- SEM of triplicate measurements from at least one of three experiments. Curves were fitted using a four-parameter sigmoidal curve fit (curves are shown on the graph; R2 = 0.99).

Figure 15. Percent inhibition of human CXCL13-mediated internalization of the human CXCR5 receptor by MAb 5378, MAb 5261, 3D2, mouse isotype control, or human isotype control.

Data points for 5261 and 5378 represent average of measurements from two independent experiments. Data points for 3D2 and Isotype Controls represent average of triplicate measurements from a single experiment. Curve was fitted using four-parameter sigmoidal curve fit (curves are shown on the graph; R2=0.99). NE=no effect.