Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

Abstract

Chronic lymphocytic leukemia is an incurable B-cell malignancy that is associated with tumor cell-mediated T-cell dysfunction. It therefore represents a challenging disease for T-cell immunotherapeutics. The CD19/CD3 bi-specific antibody construct blinatumomab (AMG103 or MT103) has been tested clinically in non-Hodgkin's lymphoma and acute lymphoblastic leukemia but has not been assessed in chronic lymphocytic leukemia. We investigated whether blinatumomab could overcome T-cell dysfunction in chronic lymphocytic leukemia in vitro. Blinatumomab was tested on peripheral blood mononuclear cells from 28 patients (treatment naïve and previously treated). T-cell activation and function, as well as cytotoxicity against leukemic tumor cells were measured. Blinatumomab induced T-cell activation, proliferation, cytokine secretion and granzyme B release in a manner similar to that occurring with stimulation with anti-CD3/anti-CD28 beads. However, only blinatumomab was able to induce tumor cell death and this was found to require blinatumomab-mediated conjugate formation between T cells and tumor cells. Cytotoxicity of tumor cells was observed at very low T-cell:tumor cell ratios. A three-dimensional model based on confocal microscopy suggested that up to 11 tumor cells could cluster round each T cell. Importantly, blinatumomab induced cytotoxicity against tumor cells in samples from both treatment-naïve and treated patients, and in the presence of co-culture pro-survival signals. The potent cytotoxic action of blinatumomab on tumor cells appears to involve conjugation of T cells with tumor cells at both the activation and effector stages. The efficacy of blinatumomab in vitro suggests that the bi-specific antibody approach may be a powerful immunotherapeutic strategy in chronic lymphocytic leukemia.

Figures

Figure 1.

Blinatumomab mediates the expansion of T cells and a loss of CLL cells in PBMC cultures. Absolute numbers of CD4+ T cells (A) and CD8+ T cells (B) were calculated from CLL PBMC cultures after 7 days in the presence or absence of blinatumomab (10 ng/mL). Columns show the mean absolute number ± SD from 15 patients [BiTE® and CD3 antibody (Ab)] and 7 patients (CD3CD28). Mean starting numbers of CD4+ and CD8+ T cells were 6.9×104 cells/mL and 4.1×104 cells/mL, respectively. UNT=untreated, BiTE®= blinatumomab (10 ng/mL), CD3 Ab= anti-CD3 antibody and CD3CD28=CD3CD28 beads. Absolute numbers of CLL cells (C) were also determined in the same cultures. For this analysis, patients were divided into treatment-naïve (•) and treated patients (⋄). Mean starting numbers of CLL cells in the treatment-naïve and treated patients were 135×104 cells/mL and 113×104 cells/mL, respectively.

Figure 2.

Blinatumomab-induced CLL cell death is maintained even in the presence of pro-survival signals. PBMC from CLL patients were incubated with blinatumomab (10 ng/mL) for 7 days in liquid culture (LC) or co-cultured on fibroblast cells transfected with CD40L, TL(CD40L). (A) The percentage of annexin V+ CLL cells after incubation in LC or on non-transfected fibroblasts (NTL) or TL(CD40L). (B) The effect of blinatumomab (10 ng/mL) on the percentage of annexin V+ CLL cells in LC or in co-culture with NTL or TL(CD40L). Columns show the mean ± SD (n=7). UNT =untreated BiTE®= blinatumomab (10 ng/mL). PBMC consisted of 87–96% CLL cells.

Figure 3.

Blinatumomab induces the proliferation of T cells in CLL patients which are predominantly of an EM phenotype. T cells from CLL PBMC cultured in the presence or absence of blinatumomab (10 ng/mL) were stained intra-cellularily for the proliferation marker Ki67. (A–B) The percentages of Ki67 expressing CD4+ (A) and CD8+ (B) T cells were measured after 3 or 7 days in culture. T-cell subsets were differentiated using CCR7 and CD45RA surface markers. T-cell subsets were defined as naive (CCR7+CD45RA+), central memory (CM) (CCR7+CD45RA−), effector memory (EM) (CCR7−CD45RA−) and highly differentiated effector memory (EMRA) (CCR7−CD45RA+). (C–F) The percentages of naive (C) and EM (D) T cells within the CD4+ compartment and EM (E) and EMRA (F) T cells within the CD8+ compartment were measured. Columns show the mean ± SD (n=7). Statistical analysis was based on the results of treated cultures compared to untreated controls at day 3 or day 7. *P<0.05), **P<0.01, ***P<0.001. UNT=untreated, BiTE®= blinatumomab (10 ng/mL), CD3CD28=CD3CD28 beads.

Figure 4.

Pro-inflammatory cytokine release from CLL patients’ PBMC after incubation with blinatumomab. (A–D) Cytokine bead array measurement of IFN-γ (A), TNF-α (B), TNFβ (C) and IL-8 (D) in the supernatant of PBMC cultures treated with blinatumomab (10 ng/mL) for 3 days. The mean concentrations are shown (n=10). (E–F) Intracellular cytokine staining for CD4+ T cells and CD8+ T cells expressing IFN-γ, TNF-α or both IFN-γ and TNF-α. Columns show the mean ± SD (n=4). Statistical analysis is based on the percentage of cytokine-positive cells in treated cultures compared to untreated controls. *P<0.05; **P<0.01; ***P<0.001.

Figure 5.

Blinatumomab increases the expression and release of cytotoxic granules from CLL patients’ T cells. T cells from CLL PBMC cultured in the presence or absence of blinatumomab (10 ng/mL) were stained for intracellular granzyme B and surface CD107 expression. The percentages of granzyme B or granzyme B and CD107 expressing CD4+ (A–B) and CD8+ (C–D) T cells after 3 days in culture were measured. The means of each group are shown (n=4). UNT=untreated, BiTE®= blinatumomab (10 ng/mL), CD3CD28=CD3CD28 beads.

Figure 6.

Visualization of CLL and T-cell clustering in the presence of blinatumomab. (A) CLL PBMC were incubated in culture for 12 h in the presence of BiTE® (10/100 ng/mL), and agonist anti-CD3 antibody (27 or 277 ng/mL) or CD3CD28 beads. T cells and CLL cells were stained with CFSE (red and green) and visualized after incubation for 12 h in the presence or absence of blinatumomab. (B) Cell cluster formations in cultures treated with blinatumomab (100 ng/mL). (C) Visualization of CLL cells (green) and a T cell (red) in a cluster by confocal microscopy. (D) Three-dimensional reconstruction of a cluster using Imaris imaging software; T cell (red) and CLL cells (green).