Afuco™ Anti-Human KMA ADCC Recombinant Antibody (MDX-1097), ADCC Enhanced (CAT#: AFC-455CL)

Figure 1 KMA is present on plasma cells from κ-isotype restricted multiple myeloma patients manifesting divergent CD45 and CD138 expression.

Bone marrow mononuclear cells were stained with anti-CD45, anti-CD138, anti-CD38 and MDX-1097 antibodies. (A) The majority of live cells (panel I: gate shown; FSC: Forward scatter; SSC: side scatter) are CD38+CD138- (panel II: right lower quadrant) and CD45+ (panel IV; right upper quadrant) while a small number of cells are CD38+CD138+CD45+ (right upper quadrant, panel II and III). Both populations are κ myeloma antigen (KMA) positive (histogram overlays on the right) as shown by a shift of MDX-1097 fluorescent peak (blue) compared to the isotype control peak (red). (B) The majority of the live cells (panel I, gate shown) are CD38+CD138− (panel II; lower right quadrant) while a distinct CD38++CD138++ is also detected (panel II; upper right quadrant). In the CD38+/CD138- population, the majority of cells are CD45+ (panel IV: upper right quadrant) while in the CD38++CD138++ the majority of cells are CD45− (panel III; lower right quadrant). Both above populations also contain CD45− and CD45+ cells (panel III: upper right quadrant and panel IV lower right quadrant). All populations detected are KMA-positive.

Asvadi, P., Cuddihy, A., Dunn, R. D., Jiang, V., Wong, M. X., Jones, D. R., ... & Spencer, A. (2015). MDX-1097 induces antibody-dependent cellular cytotoxicity against kappa multiple myeloma cells and its activity is augmented by lenalidomide. British journal of haematology, 169(3), 333-343.

Figure 2 MDX1097 has higher affinity for KMA than soluble κFLC.

Cells were stained with biotinylated MDX-1097 either alone, or in the presence of IgG/κ or κFLC, and MDX-1097 binding was quantified. Peaks: black: cells only, grey: detection antibody only, red: MDX-1097, blue: MDX-1097+IgG/κ, green: MDX-1097+κFLC.

Asvadi, P., Cuddihy, A., Dunn, R. D., Jiang, V., Wong, M. X., Jones, D. R., ... & Spencer, A. (2015). MDX-1097 induces antibody-dependent cellular cytotoxicity against kappa multiple myeloma cells and its activity is augmented by lenalidomide. British journal of haematology, 169(3), 333-343.

Figure 3 MDX-1097 mediates antibody-dependent cellular cytotoxicity of KMA-expressing κHMCL cells.

(A) Labelled JJN3 cells were treated with MDX-1097 (black bars) or isotype control (white bars) before the addition of peripheral blood mononuclear cells (PBMCs). Data represents the average of six independent experiments ± standard error of the mean. Statistical significance (P value <0·05) is represented with one asterisk. (B) Labelled JJN3 cells were incubated with different concentrations of MDX-1097 (black bars) or isotype control (white bars) before adding PBMCs at a fixed 100:1 Effector:Target ratio. Statistical significance is represented with two asterisks (P value <0·001) or one asterisk (P value <0·05). KMA, κ myeloma antigen.

Asvadi, P., Cuddihy, A., Dunn, R. D., Jiang, V., Wong, M. X., Jones, D. R., ... & Spencer, A. (2015). MDX-1097 induces antibody-dependent cellular cytotoxicity against kappa multiple myeloma cells and its activity is augmented by lenalidomide. British journal of haematology, 169(3), 333-343.

Figure 4 In vitro lenalidomide exposure enhances KMA expression levels and MDX-1097-mediated ADCC.

(A) Fold increase in the mean fluorescence intensity (MFI) of vehicle (dimethyl sulfoxide, DMSO), lenalidomide (Len), dexamethasone (Dex) or Len+Dex-treated JJN3 cells stained for KMA, CD38 and CD138 is shown. Data was collated from four independent experiments (error bars represent standard error of the mean [SEM]). Two-way analysis of variance (anova) and Bonferroni post tests were used to determine statistical significance (t-test with P value <0·05; denoted with asterisks) in expression level changes. Reduction in the level of CD138 expression post-Len and -Dex treatment was also significant (not represented on the graph). (B) Len- (black and white bars) or DMSO- (grey and hatched bars) treated JJN3 cells were used in antibody-dependent cellular cytotoxicity (ADCC) assays. Data represent the average of three independent experiments ± SEM. (C) Len- (black and white bars) or DMSO- (grey and hatched bars) treated PBMCs were used in ADCC assays.

Asvadi, P., Cuddihy, A., Dunn, R. D., Jiang, V., Wong, M. X., Jones, D. R., ... & Spencer, A. (2015). MDX-1097 induces antibody-dependent cellular cytotoxicity against kappa multiple myeloma cells and its activity is augmented by lenalidomide. British journal of haematology, 169(3), 333-343.

Figure 5 PBMCs exposed to lenalidomide in vivo induce a significantly higher level of MDX-1097-mediated antibody-dependent cellular cytotoxicity than lenalidomide-naïve PBMC.

PBMCs obtained from patients prior to (black and grey bars) or after lenalidomide treatment (white and hatched bars) were mixed at different ratios with labelled JJN3 cells coated with 200 μg/ml MDX-1097 (black and white bars) or human IgG isotype (grey and hatched bars). % JJN3 cell death was determined as described. Data represents the average of three patients ± standard error of the mean. **: P < 0·001, *: P < 0·05 as determined by 2-way analysis of variance. PMBC, peripheral blood mononuclear cells.

Asvadi, P., Cuddihy, A., Dunn, R. D., Jiang, V., Wong, M. X., Jones, D. R., ... & Spencer, A. (2015). MDX-1097 induces antibody-dependent cellular cytotoxicity against kappa multiple myeloma cells and its activity is augmented by lenalidomide. British journal of haematology, 169(3), 333-343.

Figure 6 In vitro IMiD-treated PBMCs increase MDX-1097 dependent MM cell death.

PBMCs were treated with 1 µM Lenalidomide (Len), 1 µM Pomalidomide (Pom) or vehicle (DMSO) for 72 hours prior to ADCC. MDX-1097 bound MM cells more effectively utilize IMiDtreated PBMCs to increase MM cell death compared to controls.

Figure 7 PBMCs from Lenalidomide-treated MM patients induce higher levels of MDX-1097 dependent MM cell death.

PBMCs isolated from the same patient prior to and after Len treatment (10 mg/day) were mixed with MDX-1097 or IgG spiked MM cells. MDX-1097-bound MM cells more effectively utilize the in vivo Len-treated PB immune effector cells to enhance cell death of MM cells in vitro.

Figure 8 IMiDs enhance KMA expression on MM cells and increase PB immune effector cellinduced MDX-1097 dependent MM cell killing.

Treatment of JJN3 cells with 1 µM Lenalidomide (Len) or 1 µM Pomalidomide (Pom) increases KMA expression. Blue histogram indicates isotype control. IMiD treatment also sensitises cells to enhanced PB immune effector cell-mediated ADCC in the presence of MDX-1097 compared to untreated JJN3 cells. Data shown is for a 50 to 1 PBMC:JJN3 cell ratio.

Figure 9 Lenalidomide-treated PBMCs are most effective against MDX-1097 bound, Lenalidomide-treated MM cells.

Vehicle or Len treated PBMCs and JJN3 cells were incubated together in various combinations at a fixed 50:1 PBMC:JJN3 ratio. The increased KMA levels on Len-treated JJN3 cells resulted in more MDX-1097 binding, which in turn enhanced MM cell death in the presence of Len-treated PBMCs.