Human Anti-MS4A1 Recombinant Antibody (clone OFA)

A and B, Varying amounts of C1q were added to B cells suspended in complete RPMI 1640 medium and then reacted with either Al647 OFA, Al647 RTX, or Al647 7D8-IgG4 (K322A). C, Binding of C1q to CLL cells opsonized with either OFA or with RTX, similar conditions and analyses. D-I, Cells were combined in cold medium with Al647 mAb and varying amounts of C1q and then incubated at 37°C. Aliquots were removed at the indicated times, quenched with ice-cold BSA-PBS, washed, probed with FITC anti-C1q, and then analyzed by flow cytometry for C1q binding (D-F) and mAb binding (G-I). D and G, Raji cells. E and H, Daudi cells. F and I, Z138 cells.

Pawluczkowycz, A. W., Beurskens, F. J., Beum, P. V., Lindorfer, M. A., van de Winkel, J. G., Parren, P. W., & Taylor, R. P. (2009). Binding of submaximal C1q promotes complement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20 mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher levels of CDC are induced by OFA than by RTX. The Journal of Immunology, 183(1), 749-758.

A, Fluorescence intensity of Al488 C1q and Al647 mAbs bound to Daudi cells. Very little C1q binding to RTX- or 7D8-IgG4 (K322A)-opsonized cells was observed. B and C, Fluorescence signals for Al647 OFA-opsonized samples were analyzed for colocalization with bound Al488 C1q.

Pawluczkowycz, A. W., Beurskens, F. J., Beum, P. V., Lindorfer, M. A., van de Winkel, J. G., Parren, P. W., & Taylor, R. P. (2009). Binding of submaximal C1q promotes complement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20 mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher levels of CDC are induced by OFA than by RTX. The Journal of Immunology, 183(1), 749-758.

A, Daudi cells were combined with OFA, RTX, or with no mAb in 50% C1q-depleted NHS supplemented with varying amounts of C1q. B and C, In the presence of C1q, OFA-opsonized cells are killed very rapidly. OFA-opsonized Raji (B) or Z138 (C)cells were incubated at 37°C for 1-20 min in 50% C1q-depleted NHS supplemented with 1, 2, or 35 μg/ml C1q. D and E, In C1q-depleted NHS supplemented with either 35 or 1 μg/ml C1q, binding of C1q to OFA opsonized Raji or Z138 cells is quite weak.

Pawluczkowycz, A. W., Beurskens, F. J., Beum, P. V., Lindorfer, M. A., van de Winkel, J. G., Parren, P. W., & Taylor, R. P. (2009). Binding of submaximal C1q promotes complement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20 mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher levels of CDC are induced by OFA than by RTX. The Journal of Immunology, 183(1), 749-758.

A, In medium, OFA-opsonized Raji cells bind more C1q than do RTX-opsonized cells. B and C, C3b deposition and CDC as a function of time and C1q concentration.

Pawluczkowycz, A. W., Beurskens, F. J., Beum, P. V., Lindorfer, M. A., van de Winkel, J. G., Parren, P. W., & Taylor, R. P. (2009). Binding of submaximal C1q promotes complement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20 mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher levels of CDC are induced by OFA than by RTX. The Journal of Immunology, 183(1), 749-758.

A, In medium, OFA-opsonized Daudi cells bind more C1q than do RTX-opsonized cells. B and C, C3b deposition and CDC as a function of time and C1q concentration.

Pawluczkowycz, A. W., Beurskens, F. J., Beum, P. V., Lindorfer, M. A., van de Winkel, J. G., Parren, P. W., & Taylor, R. P. (2009). Binding of submaximal C1q promotes complement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20 mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher levels of CDC are induced by OFA than by RTX. The Journal of Immunology, 183(1), 749-758.

A, In medium, OFA-opsonized Z138 cells bind more C1q than do RTX-opsonized cells. B and C, C3b deposition and CDC as a function of time and C1q concentration.

Pawluczkowycz, A. W., Beurskens, F. J., Beum, P. V., Lindorfer, M. A., van de Winkel, J. G., Parren, P. W., & Taylor, R. P. (2009). Binding of submaximal C1q promotes complement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20 mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher levels of CDC are induced by OFA than by RTX. The Journal of Immunology, 183(1), 749-758.

A-C, Dose-response profile for CLL cells from one CLL patient. D, Results for C1q binding in medium, and C3b deposition and CDC in C1qdepleted serum (35 μg/ml C1q for all cases) for CLL cells from seven patients.

Pawluczkowycz, A. W., Beurskens, F. J., Beum, P. V., Lindorfer, M. A., van de Winkel, J. G., Parren, P. W., & Taylor, R. P. (2009). Binding of submaximal C1q promotes complement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20 mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher levels of CDC are induced by OFA than by RTX. The Journal of Immunology, 183(1), 749-758.

OFA demonstrated much greater cell kill by CDC than by RTX, with 20 mg/ml eliciting high levels of cell death in all cytogenetic groups. Statistical analysis revealed that, when compared with RTX, OFA induced significantly more CDCmediated cell death in 11q - and 17p- cohorts, with borderline significant effect in the normal/13q group

Middleton, O., Cosimo, E., Dobbin, E., McCaig, A. M., Clarke, C., Brant, A. M., ... & Wheadon, H. (2015). Complement deficiencies limit CD20 monoclonal antibody treatment efficacy in CLL. Leukemia, 29(1), 107-114.

Binding of 60 nM Ofatumumab (OFA), Rituximab (RTX), or Obinutuzumab (OBI) to Daudi (A), Ramos (B), LCL1.11 (C), or P493.6 (D) cells was recorded until equilibrium was approached (not shown) followed by measurement of mAb dissociation either in plain cell culture medium or in presence of 60 nM of the respective unlabeled (unlab.) antibody. For all cell lines the dissociation of OFA was measured in presence of 180 nM unlabeled OFA (instead of 60 nM) to enhance possible cell-line differences. (E) Signal intensities were normalized to 100% at the beginning of the dissociation. The remaining signal after 1 h (black) and 2 h (gray) dissociation both in plain media (solid) and in presence of unlabeled antibody (shaded) are plotted for human B cell lines.

Bondza, S., Marosan, A., Kara, S., Lösing, J., Peipp, M., Nimmerjahn, F., ... & Lux, A. (2021). Complement-dependent activity of CD20-specific IgG correlates with bivalent antigen binding and C1q binding strength. Frontiers in immunology, 11, 609941.

Binding of 60 nM Ofatumumab (OFA), Rituximab (RTX) or Obinutuzumab (OBI) to primary human B cells isolated from blood of healthy donors was recorded until equilibrium was approached (not shown) and then mAb dissociation was measured either in plain cell culture medium or in presence of 60 nM of the respective unlabeled antibody. (A) One out of three independent measurements shown. (B) Signal intensities were normalized to 100% at the beginning of the dissociation. The remaining signal after 1 h (black) and 2 h (gray) dissociation both in plain media (solid) and in presence of unlabeled antibody (shaded) are plotted.

Bondza, S., Marosan, A., Kara, S., Lösing, J., Peipp, M., Nimmerjahn, F., ... & Lux, A. (2021). Complement-dependent activity of CD20-specific IgG correlates with bivalent antigen binding and C1q binding strength. Frontiers in immunology, 11, 609941.

OFA was infused on days 1, 8 and 29. Blood samples were obtained immediately before, and 2, 6 and 24 hrs after starting OFA infusions. Results are normalized to pre-treatment values for absolute lymphocyte counts (ALC), CD20, and CH50. Bound OFA is normalized to the 2 hr mark (first infusion), usually the maximum amount bound; values for bound C3d are normalized to maximum amount bound, observed at 2 or 6 hrs. Absolute values for these parameters are provided in Table I, along with representative uncertainties (SD). A. ALC; B. B cell surface levels of CD20; C. cell-bound OFA; D. complement titers (CH50 determinations); E. C3d deposition on B cells. The complement titer of patient N1 was low throughout the study and is not plotted in panel D. Results in B, C and E are based on duplicate determinations; CH50 titers were determined in triplicate.

Beurskens, F. J., Lindorfer, M. A., Farooqui, M., Beum, P. V., Engelberts, P., Mackus, W. J., ... & Taylor, R. P. (2012). Exhaustion of cytotoxic effector systems may limit monoclonal antibody-based immunotherapy in cancer patients. The Journal of Immunology, 188(7), 3532-3541.

These correlations showed that 8hr OFA levels were inversely correlated with pre-treatment total cell-associated CD20 and with the change in the total cellassociated CD20 between the 0 hr and 8 hr specimens.

Baig, N. A., Taylor, R. P., Lindorfer, M. A., Church, A. K., LaPlant, B. R., Pettinger, A. M., ... & Zent, C. S. (2014). Induced resistance to ofatumumab-mediated cell clearance mechanisms, including complement-dependent cytotoxicity, in chronic lymphocytic leukemia. The Journal of Immunology, 192(4), 1620-1629.

There was a progressive increase in OFA binding as the OFA concentration was increased from 0.025 μg/ml to 0.100 μg/ml.

Baig, N. A., Taylor, R. P., Lindorfer, M. A., Church, A. K., LaPlant, B. R., Pettinger, A. M., ... & Zent, C. S. (2014). Induced resistance to ofatumumab-mediated cell clearance mechanisms, including complement-dependent cytotoxicity, in chronic lymphocytic leukemia. The Journal of Immunology, 192(4), 1620-1629.