Mouse Anti-TNFRSF17 Recombinant Antibody (clone J22.9-xi) (CAT#: PABL-019)

Figure 1 J22.9-xi is a high affinity antibody for human BCMA.

A) ELISA on BCMA-coated microtiter plates and B) flow cytometry analyses using BCMA-positive MM.1S, NCIeH929, OPM-2, and RPMI-8226 cell lines. Chimeric antibodies were detected using an HRP-conjugated or PE-conjugated goat anti-human secondary antibody, respectively. Experiments were performed in sextuplicate (A) and triplicate (B), respectively. C) No binding of J22.9-xi to BCMA-negative Jurkat cells is detectable using flow cytometry. Shown is one of two independent experiments. D)Binding affinity was determined from surface plasmon resonance measurements with indicated concentrations of BCMA. Shown is one out of three independent experiments. E) Flow cytometry analysis of myeloma cells in the BM of an MM patient stained with a standard diagnostic antibody against CD138 and with J22.9-xi. Dotted lines represent isotype control staining. Shown is one representative BM sample of five analyzed samples from MM patients.

Oden, F., Marino, S. F., Brand, J., Scheu, S., Kriegel, C., Olal, D., ... & Daumke, O. (2015). Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma. Molecular oncology, 9(7), 1348-1358.

Figure 2 J22.9-xi blocks BAFF and interferes with NF-kB activation.

A) J22.9-xi blocks the interaction of BAFF with BCMA adsorbed onto microtiter plates. Error bars indicate the standard error of the mean (SEM) of triplicates (***P < 0.001, t-test); shown is one out of three independent experiments. B) NCIeH929 cells were preincubated with 10 mg J22-Xi or control-antibody for 10 min and subsequently stimulated with 500 ng APRIL for 10 or 20 min, as indicated. Cells were lysed with whole cell lysis buffer and analyzed by immunoblotting. Signals representing Phospho-IKK are indicated by an asterisk. The IKKa band serves as an internal loading control, since expression levels do not change in a stimulus dependent manner. Shown is one out of two independent experiments. C) Whole cell extracts of NCI cells treated as in (B) were used to monitor NFkB activation (EMSA). The lower band is a result of unspecific DNA binding activity within the protein extract and serves as a loading control.

Oden, F., Marino, S. F., Brand, J., Scheu, S., Kriegel, C., Olal, D., ... & Daumke, O. (2015). Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma. Molecular oncology, 9(7), 1348-1358.

Figure 3 J22.9-xi mediates strong cytotoxic effects in vitro.

A) BCMA-positive MM.1S-Luc cells were mixed with unstimulated human PBMCs (from 5 donors (#1e#5)) at an effector to target ratio of 20:1 and incubated with the indicated concentrations of J22.9-xi for 4 h (filed symbols). Open symbols indicate cytotoxic activity of J22.9-xi-N-glycan when incubated with PBMCs from donor 1 or 2. Cytotoxicity was calculated relative to isoAb (dotted line) and subtracted from 100 using bioluminescence measurements of the living cells. B) No detectable difference in binding to MM.1S cells is observed between J22.9-xi and J22.9-xi-N-glycan using flow cytometry. Shown is one out of two independent experiments.

Oden, F., Marino, S. F., Brand, J., Scheu, S., Kriegel, C., Olal, D., ... & Daumke, O. (2015). Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma. Molecular oncology, 9(7), 1348-1358.

Figure 4 J22.9-xi is effective against tumors in a xenografted mouse model.

A) Experimental timeline: antibody treatment of xenografted SCID Beige mice for the first 5 days starting with the day of tumor cell challenge (1×107 cells/mouse). Open circles indicate days of bioluminescence measurements. B) Course of tumor growth in mice treated with 2 (n=5), 20 (n=5) or 200 ug (n=4) of J22.9-xi or 200 ug of either isoAb (n=5) or J22.9-xi-N-glycan (n=6), and without tumor (n=3; control). Background of bioluminescence was determined using NSG mice not challenged with MM.1S-Luc cells (n=3; control). C) Tumor development over time, represented as area under curve of (B), between days 9 and 44. Mean of total flux from unchallenged mice was subtracted. Shown are the mean values with SEM (*P<0.05, 2-tailed t-test). D) Survival of antibody-treated and control xenografted SCID Beige mice. The P values were calculated using the Log-rank (ManteleCox) Test. E) Distribution of MM.1S-Luc cells in the indicated groups at day 44 post tumor challenge; dorsal view.

Oden, F., Marino, S. F., Brand, J., Scheu, S., Kriegel, C., Olal, D., ... & Daumke, O. (2015). Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma. Molecular oncology, 9(7), 1348-1358.