Figure 2.
The effect of glofitamab and HPK1 inhibitors on immune synapse formation, granzyme B induction, and CLL cell death. (A-B) CLL cells were seeded on poly-L-lysine coated glass, then treated with 300 nM HY-138568 or with 600 nM BGB15025 for 2 hours. Then the cells were incubated with 1 μg/mL of the CD20XCD3 bispecific antibody glofitamab or human immunoglobulin G (IgG) kappa isotype control for 48 hours. The cells were fixed and permeabilized, followed by staining for CD8 (white), granzyme B (red), immunoglobulin M (IgM) (green), and DAPI (4′,6-diamidino-2-phenylindole) (blue). The images presented are 3D reconstruction of representative confocal imaging of synapse formation between B (green) and CD8+ T (white) CLL cells. Patients are coded by numbers and displayed as CLL_number. (A) Representative images of cells treated with isotype control or glofitamab. (B) Representative images of cells treated with isotype control, glofitamab, glofitamab and HY-138568, or glofitamab and BGB15025 (n = 5). (C-E) B and T cells were isolated from CLL patients–derived PBMCs using B-CLL and pan T isolation kits. Isolated B cells (CLL cells) were labeled with BioTracker 488 Green carboxyfluorescein succinimidyl ester (CFSE) dye. Isolated T cells were treated with 300 nM HY-138568 or with 600 nM BGB15025 for 2 hours. Then, CLL cells and autologous T cells were seeded at 4:1 effector to target ratio and incubated with 1μg/mL of the anti-CD20 × anti-CD3 bispecific antibody glofitamab or isotype control for 24 hours. Cytotox red dye was used to detect cell killing. CLL cell killing (% green + red cells/green cells) was analyzed using Incucyte system for indicated times. At least 4 images from distinct regions within each well were taken at intervals of 6 hours. The experiment was performed in triplicates. (C) Representative images from 2 time points using cells from 1 patient. (D) Quantification of % CLL cell killing using glofitamab (± HPK1 inhibitors) or isotype control after 24 hours of incubation (n = 8). (E) SLD (in micrometers) of clusters after 24 hours of incubation. (F) CLL cells were treated with the indicated concentration of HY-138568 or BGB15025 for 2 hours. Following treatment, the cells were incubated with 1 μg/mL of the anti-CD20 × anti-CD3 bispecific antibody glofitamab or isotype control for 48 hours. Granzyme B levels were measured from supernatants using ELISA-based assay platform (n = 6). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. HY, HY-138568; NT, no treatment; SLD, sum of longest diameter.

The effect of glofitamab and HPK1 inhibitors on immune synapse formation, granzyme B induction, and CLL cell death. (A-B) CLL cells were seeded on poly-L-lysine coated glass, then treated with 300 nM HY-138568 or with 600 nM BGB15025 for 2 hours. Then the cells were incubated with 1 μg/mL of the CD20XCD3 bispecific antibody glofitamab or human immunoglobulin G (IgG) kappa isotype control for 48 hours. The cells were fixed and permeabilized, followed by staining for CD8 (white), granzyme B (red), immunoglobulin M (IgM) (green), and DAPI (4′,6-diamidino-2-phenylindole) (blue). The images presented are 3D reconstruction of representative confocal imaging of synapse formation between B (green) and CD8+ T (white) CLL cells. Patients are coded by numbers and displayed as CLL_number. (A) Representative images of cells treated with isotype control or glofitamab. (B) Representative images of cells treated with isotype control, glofitamab, glofitamab and HY-138568, or glofitamab and BGB15025 (n = 5). (C-E) B and T cells were isolated from CLL patients–derived PBMCs using B-CLL and pan T isolation kits. Isolated B cells (CLL cells) were labeled with BioTracker 488 Green carboxyfluorescein succinimidyl ester (CFSE) dye. Isolated T cells were treated with 300 nM HY-138568 or with 600 nM BGB15025 for 2 hours. Then, CLL cells and autologous T cells were seeded at 4:1 effector to target ratio and incubated with 1μg/mL of the anti-CD20 × anti-CD3 bispecific antibody glofitamab or isotype control for 24 hours. Cytotox red dye was used to detect cell killing. CLL cell killing (% green + red cells/green cells) was analyzed using Incucyte system for indicated times. At least 4 images from distinct regions within each well were taken at intervals of 6 hours. The experiment was performed in triplicates. (C) Representative images from 2 time points using cells from 1 patient. (D) Quantification of % CLL cell killing using glofitamab (± HPK1 inhibitors) or isotype control after 24 hours of incubation (n = 8). (E) SLD (in micrometers) of clusters after 24 hours of incubation. (F) CLL cells were treated with the indicated concentration of HY-138568 or BGB15025 for 2 hours. Following treatment, the cells were incubated with 1 μg/mL of the anti-CD20 × anti-CD3 bispecific antibody glofitamab or isotype control for 48 hours. Granzyme B levels were measured from supernatants using ELISA-based assay platform (n = 6). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. HY, HY-138568; NT, no treatment; SLD, sum of longest diameter.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal