SEATTLEXcyte Therapies is testing in preclinical studies the feasibility of a new technology that would take T cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) and spur them into action with key antibodies, with the goal of reinfusing them into the patient ready to fight B-cell tumors. Mark L. Bonyhadi, PhD, of Xcyte Therapies in Seattle, Washington, reported that the company’s Xcellerate technology "produces activated T cells that can interact with leukemic B cells and alter their behavior in vitro. We hope this will be followed by apoptosis or clearance of B cells in B-CLL patients."
Dr. Bonyhadi explained that B-CLL is characterized by the proliferation and accumulation of mature-appearing, but biologically immature B cells that are resistant to normal programmed cell death signals. T cells in B-CLL patients often display an activated phenotype but decreased mitogen and allogeneic response, indicative of T-cell dysfunction. "In CLL there is a vast abundance of B cells, while the percentage of T cells relative to leukemic cells is quite low, around 1%-10%," he said.
CLL patients also may have reduced T cell receptor (TCR) z chain expression, low CD28 and CD40L expression, and impaired delayed hypersensitivity responses. "Furthermore, many current CLL treatments disable the T-cell arm of the immune response," Dr. Bonyhadi added.
Activating T Cells
The new method will be tested in future clinical trials aimed at preventing infection and promoting anti-leukemic responses through use of artificial antigen-presenting cells to activate autologous T cells that are then reinfused into the patient.
"The antibodies are attached to small paramagnetic beads about half the size of a T cell," Dr. Bonyhadi said. "The effect is polyclonal and activates all T cells. We were concerned that our beads might not be able to find the few T cells in blood from these patients, but we were pleasantly surprised."
The ex vivo activation and expansion of T cells uses immobilized anti-CD3 and anti-CD28 antibodies on magnetic beads. Normal donor T cells undergoing the process show increased expression of CD40 ligand, IL-2 receptor, 41BB, IL-2, and IFNg. The treated cells also exhibit effector cell responses when restimulated with antigen.
"The beads mimic antigen-presenting cells by providing T cells unopposed signals 1 and 2 required for activation," Dr. Bonyhadi said.
In B-CLL patients, few of the circulating peripheral blood mononuclear cells (PBMC) are of the T cell lineage and these often display functional defects.
Dr. Bonyhadi said that preliminary in vitro preclinical studies of PBMC from B-CLL patients treated with this approach showed 100-1,000 fold expansion of T cells over 12-16 days, even in patients who have received prior treatment such as fludarabine (Fludara). At the end of culture, the cell product contained few B cells and consisted mostly of T cells that exhibited normal functions. These treated T cells also expressed high levels of IFNg, CD40 ligand, 41BB, OX40, I-CAM, and IL-2 receptor. When reactivated, these cells displayed effector T-cell characteristics.
Up-regulation of Receptors
‘‘Both CD4+ and CD8+ T cells are maintained during the expansion phase of the patient’s PBMC, and we see up-regulation of key receptors and activation markers during culture and expansion, similar to levels seen in blood from normal donors," Dr. Bonyhadi said. "In particular, upregulation of IFNg and other cytokines is characteristic of effector cells."
Recently, in preclinical studies, PBMC from B-CLL patients were processed at full scale. Starting cultures contained > 85% B cells and < 4% T cells. T cells expanded over 100 fold in 16 days and were of both the CD4+ and CD8+ phenotype. The end cell product contained < 1% B cells. The T cells exhibited functional traits similar to those observed for normal donors. Moreover, the decrease in TCR expression observed in freshly isolated B-CLL patient T cells was corrected following in vitro treatment, yielding T cells that expressed surface TCR at normal levels and were reflective of a diverse repertoire.
The activated T cells can also upregulate I-CAM and FAS expression on leukemic B cells in culture, which nontreated T cells did not do. "This demonstrates that in the preclinical in vitro setting these activated autologous T cells are capable of interacting with the B-CLL patient’s leukemic B cells and of changing B-cell behavior. This approach may play a role in adoptive immunotherapy for CLL patients." He said that the investigators envision harvesting T cells before patients begin treatment with fludarabine or irinotecan(Drug information on irinotecan) (Camptosar), then activating, expanding, and reinfusing the T cells following chemotherapy.