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

Abstract

© F e r r a t a S t o r t i F o u n d a t i o n 2 0 1 3of CLL activation and pro-survival signals. Given these data, this approach represents a novel and promising therapeutic strategy for the treatment of CLL and, potentially, other lymphoid malignancies. Methods Blood samples from patients with chronic lymphocytic leukemiaTwenty-eight CLL patients (aged 53-83 years) were studied with full ethical approval. All blood samples from patients were obtained with informed consent (see Online Supplementary Methods). The majority of the assays were performed on fresh samples from treatment-naïve patients, except those for which the results are shown in Figure 1: in these assays, samples from patients who had received standard chemotherapy treatment were also tested. Antibodies and flow cytometryA full list of the antibodies used can be found in the Online Supplementary Methods. Blinatumomab (MT103, AMG103) was provided by Amgen Inc. (Munich, Germany). T cells were identified using CD4 and CD8 markers, while CLL cells were CD5 + CD4-CD8 -(>99% CD19 + ). Cell culturesPeripheral blood mononuclear cells (PBMC) were incubated in RPMI 1640 supplemented with 5% AB serum (AB media) for 3-7 days. Blinatumomab was added to PBMC cultures at a concentration of 10 or 100 ng/mL. Human T-cell activator CD3/CD28 dynabeads (Invitrogen) were added at 1x10 5 beads/1x10 6 PBMC as a positive control for T-cell activation. For absolute counts of T cells and CLL cells, an anti-CD3 antibody (Invitrogen) at a dose of 27.7 or 277 ng/mL was used as a positive control (same molar concentration as 10 or 100 ng/mL blinatumomab). Intracellular Ki67 stainingPBMC were labeled with antibodies against CD4, CD8, CCR7 and CD45RA, fixed and permeabilized (Fix and Perm with 1% NP40, Caltag-Medsystems, Buckingham, UK) before staining with a Ki67-FITC antibody for flow cytometric analysis. Cytokine secretion assayCytokines in tissue culture supernatants were measured using a Human Th1/Th2 11plex RTU Flowcytomix kit (eBioscience) for simultaneous detection of 11 cytokines. Intracellular cytokine staining and the determination of CD107 and granzyme B expressionPBMC were cultured (3 days) with 10 ng/mL blinatumomab, before treatment with Golgi plug and Golgi stop (BD biosciences), and incubation with an anti-CD107 antibody (5 h at 37°C). Intracellular expression of granzyme B or interferon (IFN)-g and tumor necrosis factor (TNF)-α in CD4 or CD8 T cells was measured by flow cytometry. Absolute counts of T cells and chronic lymphocytic leukemia cellsPBMC samples that had or had not been treated with blinatumomab (7 days) were surface-stained with antibodies against annexin V, CD5, CD8 and CD4. Absolute counts were determined by flow cytometry using Cytocount beads (Dako, Stockport, UK). Flow cytometry-based cytotoxicity assayMouse fibroblast cells transfected with CD40L [TL(CD40L)] and non-transfected cells (NTL) were cultured as previously described. 21 For co-cultures, irradiated (80 Gy) NTL or TL(CD40L) cells were seeded into a 48-well plate, and allow...

A Novel Method Using Blinatumomab for Efficient, Clinical-Grade Expansion of Polyclonal T Cells for Adoptive Immunotherapy

“…Final products had mostly a central memory and effective memory phenotype within both helper and cytotoxic T cell subsets. These results are consistent with previous reports showing induction of effector memory CD4 + and CD8 + T cells following blinatumomab treatment in vitro and in vivo (18,31,32). Interestingly, BET cells expressed CD27, the receptor for CD70, in .70% of CD4 + and CD8 + subsets.…”

“…To expand T cells, freshly isolated PBMC from untreated CLL patients or normal donors were plated at 3 3 10 6 /ml in serum-free X-VIVO 15 medium (Lonza) containing 0.1 mM gentamicin (Fisiopharma, Palomonte, Italy), 10 ng/ml blinatumomab (AMG103; Amgen) (17,18), and 500 IU/ml rhIL-2 (Bayer HealthCare Pharmaceuticals, Berlin, Germany). In two CLL cases, frozen PBMC were used.…”

MGD011, A CD19 x CD3 Dual-Affinity Retargeting Bi-specific Molecule Incorporating Extended Circulating Half-life for the Treatment of B-Cell Malignancies

“…MGD011, however, was able to eliminate leukemic cells in CLL patient samples by engaging and expanding the patient's residual T cells in vitro from a low E:T cell ratio to one exceeding the target cell population. These data also confirm that CLL T cells can be redirected to become cytolytic against leukemic cells in the presence of MGD011, as previously reported for blinatumomab (19), even though T-cell dysfunction in CLL, such as defects in immune synapse formation, co-stimulatory/accessory molecule expression, and cytokine release have been reported (20)(21)(22).…”

Blinatumomab: First Global Approval

“…In a study using peripheral blood mononuclear cells from either treatment-naïve or previously-treated patients with chronic lymphocytic leukaemia (CLL), blinatumomab induced T-cell proliferation and activation, cytokine secretion and granzyme B release, and induced tumour cell death at very low T-cell: tumour cell ratios [21]. These findings suggest that blinatumomab may be of use in overcoming immunodeficiency in the treatment of CLL [21].…”

“…These findings suggest that blinatumomab may be of use in overcoming immunodeficiency in the treatment of CLL [21]. Blinatumomab may also have therapeutic potential in acute myeloid leukaemia (AML), as in blast cell cultures from patients with AML, the presence of blinatumomab resulted in a decrease in CD33?…”