Article Figures & Data
Figures
- Figure 1.
Selection of CD40-specific VHHs. A, CD40 expression on wild-type (WT; gray, filled histogram) and CD40-transfected (black, open histogram) HEK293T cells. B, CD40 specificity of VHH binding. WT and CD40-transfected HEK293T cells were incubated with V12t, V15t, V19t (1 μmol/L), or medium control, and VHH binding was measured by flow-cytometric detection of the Myc tag. Representative histograms of three experiments. gMFI, geometric mean fluorescence intensity. C, CD40 expression on primary CLL cells. Representative histograms of five experiments. D, Binding of CD40-specific VHHs (1 μmol/L) to primary CLL cells as in B (n = 5). E, Primary CLL PBMCs were cultured with V12t, V15t, V19t (all 1 μmol/L), recombinant multimeric CD40L (100 ng/mL), or medium control and analyzed for CD86 and CD95 expression after 48 hours (n = 5). F, Primary CLL PBMCs were preincubated with V12t, V15t, V19t (all 1 μmol/L), or medium control for 30 minutes, and then cultured with recombinant multimeric CD40L (100 ng/mL) for 48 hours and analyzed for CD86 and CD95 expression after 48 hours (n = 9). Data, mean and SEM. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. For D, repeated-measures one-way ANOVA followed by Dunnett post hoc test compared with no VHH; for E and F, one-way ANOVA followed by Dunnett post hoc test compared with medium control.
- Figure 2.
The bispecific anti-CD40-Vδ2 VHHs induce target cell lysis. A, Binding of bispecific anti-CD40-Vδ2 VHHs. CD40-transfected or wild-type (WT) HEK293T cells were incubated with V12-5C8t, V15-5C8t, V19-5C8t (1 μmol/L), or medium control, and VHH binding was measured by flow-cytometric detection of the Myc tag. Representative histograms of two experiments. B, Specific lysis of the CII cell line after overnight culture with healthy donor–derived Vγ9Vδ2 T cells in a 1:2 effector:target ratio in the presence of the indicated concentrations of the bispecific anti-CD40-Vδ2 VHHs V12-5C8t, V15-5C8t, or V19-5C8t (n = 5). bsVHH, bispecific VHH. C, Specific lysis of primary CLL cells after overnight culture with healthy donor–derived Vγ9Vδ2 T cells in a 1:1 ratio in the presence of the indicated concentrations of the bispecific anti-CD40-Vδ2 VHHs V12-5C8t, V15-5C8t, or V19-5C8t (n = 3). Specific lysis was calculated by correcting for background cell death in conditions without Vγ9Vδ2 T cells. Data, mean and range, with the line depicting a nonlinear regression curve. B and C, Nonlinear regression analysis; “0” value entered as 10−4.
- Figure 3.
The bispecific anti-CD40-Vδ2 VHH has a high binding affinity and potently activates Vγ9Vδ2 T cells. A, Binding of the bispecific anti-CD40-Vδ2 VHH to Vγ9Vδ2 T cells and CD40+ cells. Healthy donor–derived Vγ9Vδ2 T cells (green, n = 3 donors) or CII cells (black, triplicate) were incubated with the indicated concentrations of V19-5C8, and VHH binding was measured by flow-cytometric detection of anti-llama IgG heavy- and light-chain antibodies; geometric mean fluorescence plotted. bsVHH, bispecific VHH; gMFI, geometric mean fluorescence intensity. B, Activation of Vγ9Vδ2 T cells by the bispecific anti-CD40-Vδ2 VHH. Healthy donor–derived Vγ9Vδ2 T cells and CII target cells were cultured in a 1:1 ratio with V19-5C8 for 4 hours in the presence of Brefeldin A, GolgiStop, and anti-CD107a to measure IFNγ, TNF, and IL2 production and degranulation by flow cytometry (n = 3). C, Specific lysis of CII or MM.1s target cells after overnight culture with healthy donor–derived Vγ9Vδ2 T cells in a 1:1 ratio in the presence of V19-5C8 (circles) or the irrelevant bispecific anti-CD1d-Vδ2 VHH 1d7-5C8 (squares; all n = 4). Specific lysis was calculated by correcting for background cell death in conditions with Vγ9Vδ2 T cells, without bispecific VHH. D, Specific lysis of wild-type (WT) or CD40-transfected HEK293T cells as in C (n = 4). Specific lysis was calculated by correcting for background cell death in conditions without Vγ9Vδ2 T cells. Data, mean and range, with the line depicting a nonlinear regression curve (A–C) or mean and SEM (D). ****, P < 0.0001. For A–C, nonlinear regression analysis; for D, mixed-effects analysis with Šidák post hoc test comparing CD40-transfected versus WT.
- Figure 4.
The bispecific anti-CD40-Vδ2 VHH activates Vγ9Vδ2 T cells from CLL patients and induces autologous tumor lysis. A, Specific lysis of primary CLL cells after overnight culture with healthy donor–derived Vγ9Vδ2 T cells in a 1:1 ratio in the presence of V19-5C8 (n = 10). B, CLL PBMCs were cultured on irradiated 3T3 or CD40L+ 3T40L fibroblasts for 72 hours. Specific cell death of harvested CLL PBMCs after subsequent overnight culture with healthy donor–derived Vγ9Vδ2 T cells (1:1 ratio), healthy donor–derived Vγ9Vδ2 T cells and V19-5C8 (1:1 ratio, 100 nmol/L), venetoclax (10 nmol/L), or medium control (n = 3). C–E, CLL PBMCs were preincubated with 10, 100, or 1,000 nmol/L V19-5C8 or medium control for 30 minutes and then cultured in the presence of recombinant multimeric CD40L (100 ng/mL) for 48 hours. C, CD86 and CD95 expression after 48 hours (n = 6). D, Specific cell death after subsequent culture with the indicated concentrations of venetoclax for 24 hours (n = 6). E, Bcl-xL expression after 48 hours (n = 3). F and G, PBMCs from CLL patients were enriched for T cells by depletion of CD19+ CLL cells and then cultured with CD19+ CLL cells in a 1:1 ratio with V19-5C8 (10 nmol/L), aminobisphosphonates (ABP; 25 μmol/L pamidronate), or medium control in the presence of Brefeldin A, GolgiStop, and anti-CD107a to measure IFNγ production (F) and degranulation (G) by flow cytometry (n = 7). H, Lysis of primary CLL cells by autologous Vγ9Vδ2 T cells. CD3+ cells were isolated from CLL PBMCs and cultured with total CLL PBMCs in a 5:1 (CD3:PBMC; ±1:20 Vδ2:PBMC ratio; range, 1:10–1:24) or 20:1 (CD3:PBMC; ±1:3 Vδ2:PBMC ratio; range, 1:3–1:6) ratio with V19-5C8 (10 nmol/L) or medium control. Live CLL cells were quantified by flow cytometry using counting beads (n = 5). I, Percentage Vγ9Vδ2 T cells after PBMCs were cultured with IL2 (50 IU/mL) or IL2 and the bispecific VHH V19-5C8 (10 nmol/L) for 1 week (n = 5). Data, mean and SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For A, C, and E–H, repeated-measures one-way ANOVA followed by Dunnett post hoc test compared with conditions without VHH; for B, two-way ANOVA followed by Šidák post hoc test comparing each treatment condition between the CLL groups; for D, two-way ANOVA followed by Dunnett post hoc test comparing conditions to medium control; and for I, paired t test.
- Figure 5.
The bispecific anti-CD40-Vδ2 VHH is active against primary MM. A, CD40 expression on primary MM cells. Representative histogram of four donors tested. B, BM of MM patients was cultured overnight in the presence or absence of healthy donor–derived Vγ9Vδ2 T cells in a 1:1 (Vγ9Vδ2-T:plasma cell) ratio in the presence of the bispecific VHH V19-5C8 (10 pmol/L or 10 nmol/L). Live plasma cells were quantified by flow cytometry using counting beads (n = 5). C and D, Mononuclear cells from the BM of MM patients were cultured overnight with V19-5C8 (10 nmol/L), aminobisphosphonate (ABP; 10 μmol/L zoledronic acid), or medium control in the presence of Brefeldin A, GolgiStop, and anti-CD107a to measure cytokine production (C) and degranulation (D) by flow cytometry (n = 6). Data, mean and SEM. *, P < 0.05; **, P < 0.01. B–D, Repeated-measures one-way ANOVA followed by Dunnett post hoc test compared with conditions without VHH.
- Figure 6.
The bispecific anti-CD40-Vδ2 VHH prolongs survival in vivo. Immunodeficient mice were irradiated on day −1 and grafted (i.v.) with 2.5 × 106 MM.1s cells on day 0. Mice received PBS or human Vγ9Vδ2 T cells (1 × 107 cells; i.v.) on days 7, 14, and 21, followed by PBS or V19-5C8 (5 μg per mouse; i.p.) twice weekly starting on day 9. A, Schematic overview of treatment schedule. B, Kaplan–Meier analyses of mouse survival (control: n = 6; bispecific VHH: n = 6; Vγ9Vδ2 T cells: n = 8; bispecific VHH + Vγ9Vδ2 T cells: n = 8). C, Body weight after 7 weeks of treatment relative to individual body weight at time of tumor injection. **, P < 0.01. Data, mean and SD. B, Mantel–Cox log-rank test followed by Holm–Šidák post hoc test.
Tables
- Table 1.
CLL patient characteristics.
N 44 Age in years 69.5 (range, 39–89) Sex, % female 36.4 IGHV, % M-CLL 55.2 (for 29 known) Rai, % stage 0 67.9 (for 28 known) ALC × 109/L 66.0 (13.1–422) Note: Data presented as a percentage or median (range).
Abbreviations: ALC, absolute leukocyte count; M-CLL, CLL cells with a mutated IGHV status.
Supplementary Data
- Supplementary Figures - Supplementary Figures
- Supplementary Tables and Figure Legends - Supplementary Tables 1-2 and Figure legends
Volume 9, Issue 1
