Mouse Anti-TNFRSF17 Recombinant Antibody (clone CA8) (CAT#: HPAB-0009-DJB)

Figure 1 FMAT Binding Assay-Figure showing the results of the FMAT assay for CA8 antibody binding to human and cyno BCMA expressing HEK293 cells. Human chimeric CA8 binds well to human and cyno BCMA expressing cells.

Figure 2 ELISA Binding Assay-Figure showing the ELISA results for CA8 antibodies binding to human and cyno BCMA recombinant proteins. This clearly shows that human chimeric CA8 antibodies bind to human and cyno BCMA proteins equally.

Figure 3 BiaCore Binding Assay-Figure showing the binding of CA8 to BCMA-Fc, TACI-Fc and BAFF-R-Fc proteins in the Biacore experiment.

CA8 chimera antibody does not bind to TACI or BAFF-R proteins.

Figure 4 Cell binding assay-Figure showing binding of chimeric CA8 to a panel of multiple myeloma cell lines as determined by FACS.

Binding to H929, OPM-2, JJN-3 and U266 was tested by flow cytometry and mean fluorescence intensity (MFI) values measured to determine binding. Synagis was used as an irrelevant isotype control.

Figure 5 Viability assay dose response curves-Figure showing dose response curves for the unconjugated (Naked) and vcMMAE and mcMMAF antibody-drug conjugates of chimeric CA8 or humanized J6M0 antibodies.

Antibody drug conjugates were tested against human multiple myeloma cell lines NCI-H929 and OPM2.

Figure 6 Impact of chimeric CA8 on Annexin-V.

Chimeric CA8 ADC treatment results in increased Annexin-V staining of NCI-H929 cells.
(A) Histograms of Annexin-V-FITC (FL1-H; top panels) and Live cell propidium iodide staining (FL3-H; bottom panels) after treatment with increasing concentrations of chimeric CA8 ADCs (B) Quantification of Annexin-V positive NCI-H929 cells after a 96 hour treatment with the indicated concentrations of chimeric CA8 ADCs. Pactitaxel (100nM) was used as a positive control for apoptosis and control chimera lgG1 was used as a negative control. Cells were seeded in 12-well plates (2x105 cells per well in 1 mL of RPMI + 10% FBS). Antibody or ADC was added 6 hours after cell seeding.

Figure 7 Impact of CA8 chimeric antibody on cell cycle.

Cell cycle histograms of NCI-H929 cells treated with unconjugated chimeric CA8, chimeric CA8-vcMMAE ADC or chimeric CA8-mcMMAF ADC at 50 ng/ml for the timepoints indicated. Pactitaxel (100nM) was used as a positive control for G2/M cell cycle arrest and cell death. Control human lgG1 was used as a negative control. Cell cycle analysis was carried out at the times shown on the graphs.

Figure 8 Impact of CA8 chimeric antibody on cell cycle.

Quantification of the 4N DNA cell population indicative of G2/M arrest and sub-2N DNA cell population indicative of cell death for each of the treatments indicated. Cells were seeded in 12-well plates (2x10⁵ cells per well in 1 ml_ of RPMI + 10% FBS). Antibody or ADC was added 6 hours after cell seeding.

Figure 9 Viability assay dose response curves.

Figure showing dose response curves in a cell viability assay for chimeric CA8 antibody, chimeric CA8-vcMMAE and chimeric CA8-mcMMAF antibody-drug conjugates in human multiple myeloma cell lines (A) NCI-H929 (B) U266-B1 (C) JJN3 and (D) OPM2. Antibody was added to the cells and the number of viable cells after 96 hours measured using CelltiterGlo.Data points represent the mean of triplicate CellTiterGlo measurements. Error bars represent standard error.

Figure 10 ADCC assay-Figure showing ADCC activity of chimeric CA8 and defucosylated (Fc enhanced) CA8 with target cells expressing BCMA.

Human NK cells were incubated with europium labelled ARH77 10B5 BCMA transfected target cells in the presence of varying concentrations of antibody. Europium release from the target cells was measured and specific lysis calculated. (A) ADCC dose response curves of chimeric CA8 compared to isotype control. (B) ADCC dose response curves for chimeric CA8 and defucosylated chimeric CA8 (Fc enhanced), against the BCMA expressing cell line ARH77 10B5.