Anti-CD20 monoclonal antibody with enhanced affinity for CD16 activates NK cells at lower concentrations and more effectively than rituximab

Abstract

Growing evidence indicates that the affinity of monoclonal antibodies (mAbs) for CD16 (FcgammaRIII) plays a central role in the ability of the mAb to mediate antitumor activity. We evaluated how CD16 polymorphisms, and mAb with modified affinity for target antigen and CD16, affect natural killer (NK) cell phenotype when CD20(+) malignant B cells were also present. The mAb consisted of rituximab (R), anti-CD20 with enhanced affinity for CD20 (AME-B), and anti-CD20 with enhanced affinity for both CD20 and CD16 (AME-D). Higher concentrations of mAb were needed to induce CD16 modulation, CD54 up-regulation, and antibody-dependent cellular cytotoxicity (ADCC) on NK cells from subjects with the lower affinity CD16 polymorphism. The dose of mAb needed to induce NK activation was lower with AME-D irrespective of CD16 polymorphism. At saturating mAb concentrations, peak NK activation was greater for AME-D. Similar results were found with measurement of CD16 modulation, CD54 up-regulation, and ADCC. These data demonstrate that cells coated with mAb with enhanced affinity for CD16 are more effective at activating NK cells at both low and saturating mAb concentrations irrespective of CD16 polymorphism, and they provide further evidence for the clinical development of such mAbs with the goal of improving clinical response to mAb.

Figure 1.

Anti-CD20 mAbs. Schematic illustrating AME-B with modifications to enhance affinity to CD20 and AME-D with modifications to enhance affinity to both CD20 and CD16.

Figure 2.

Modulation of NK cell CD16. CD16 expression of NK cells as measured by CD16 median fluorescence (n = 12). (A) Dose response curves for different mAbs. Error bars indicate SEM. (B) EC₅₀ values for different mAbs. Loss of surface CD16 occurred at lower mAb concentrations with AME-D. Horizontal lines represent the mean.

Figure 3.

Effect of mAb and CD16 polymorphism on modulation of CD16. The mAb EC₅₀ as measured by decrease in CD16 expression was determined on NK cells from 12 subjects with various CD16 polymorphisms (n = 4 VV, 4 VF, 4 FF). (A) EC₅₀ values for AME-B, AME-D, and R grouped by polymorphism. (B) The same data with EC₅₀ values for FF, VF, and VV grouped by mAb. Horizontal lines represent the mean.

Figure 4.

Up-regulation of NK cell CD54. Up-regulation of CD54 expression as measured by the percentage of CD54_bright NK cells (n = 12). (A) Dose response curves for different mAbs. Error bars indicate SEM. (B) EC₅₀ values for different mAbs. Up-regulation of surface CD54 occurred at lower mAb concentrations with AME-D. Horizontal lines represent the mean.

Figure 5.

Effect of mAb and CD16 polymorphism on up-regulation of CD54. The mAb EC₅₀ as measured by an increase in the number of CD54_bright NK cells was determined on cells from 12 subjects with various CD16 polymorphisms (n = 4 VV, 4 VF, 4 FF). (A) EC₅₀ values for AME-B, AME-D, and R grouped by polymorphism. (B) The same data with EC₅₀ values for FF, VF, and VV grouped by mAb. Horizontal lines represent the mean.

Figure 6.

Peak activation of NK cells. The percentage of NK cells activated (as indicated by CD54_bright expression) was identified for each mAb at peak activation irrespective of mAb concentration. Peak CD54 activation for AME-B or AME-D is compared with that of R. NK cell activation by mAb-coated target cells was greater for AME-D than for AME-B or R in each sample tested. (Top) AME-B versus rituximab. (Bottom) AME-D versus rituximab.

Figure 7.

Comparison of CD16 modulation, CD54 activation, and ADCC. The EC₅₀ for AME-D and R was evaluated for CD16 modulation (A), CD54 up-regulation (B), and ADCC (C) and plotted on a log scale and grouped by polymorphism (n = 4 VV, 4 VF, and 4 FF for each assay). Similar results were found whether the readout was loss of surface CD16, NK activation as determined by CD54 up-regulation, or lysis of target cells as determined by ADCC. Error bars indicate SEM.

Figure 8.

Inhibition of Raji xenograft growth by AME-D and R. AME-D and R were compared for their ability to inhibit growth of Raji xenografts in SCID mice. Mice were inoculated with tumor cells on both flanks and treated 3 days later with the indicated dose of AME-D or R. The size of tumors 31 days after tumor inoculation was measured. Horizontal lines represent the mean.