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Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is a key negative regulator of T cell activation and proliferation. Ipilimumab is a human monoclonal antibody that specifically blocks the binding of CTLA-4 to its ligand. To test the hypothesis that blockade of CTLA-4 by ipilimumab could augment graft-versus-malignancy effects (GVM) without a significant impact on graft-versus-host disease (GVHD), we conducted a phase I clinical trial of ipilimumab infusion in patients with relapsed malignancy following allogeneic hematopoietic stem cell transplant (allo-HSCT). Here we report the analysis of peripheral blood T lymphocyte reconstitution, T regulatory cell (Treg) expression and T cell activation markers after a single dose of ipilimumab in 29 patients. Peripheral blood samples were collected from all patients before and after ipilimumab infusion. We analyzed lymphocyte immunophenotyes, including levels of CD4+CD25high cells and T cell activation markers in all cases. Levels of CD4+CD25highFoxp3+ cells and intracellular CTLA-4 in CD4+ T cells were also assessed in the last 11 cases. We found that baseline levels of CD4 and CD45RO positive T cells were lower in patients compared to normal controls. More than 50% patients had abnormally low lymphocyte counts, either CD4 or/and CD8 T cells, and some had no circulating B lymphocyte. The percentages of both CD4+CD25high and CD4+CD25highFoxp3+ T cells were significantly higher in patients prior to ipilimumab infusion than in healthy donors. 20 of 29 patients showed an elevated level of CD4+CD25low activated T cells at baseline while only 3 of 26 healthy donors had such a population of activated T cells. After ipilimumab infusion, both CD4+ and CD8+ T lymphocyte counts significantly increased. There was no consistent change in absolute lymphocyte count, or in T cells expressing the activation marker CD69. However, CD4+CD25low T cells in 20 of 29 patients, and CD4+HLA-DR+ T cell in the last 10 patients increased in the first 60 days following ipilimumab infusion. Although the percentages of both CD4+CD25high and CD4+CD25highFoxp3+ T cells significantly decreased during the observation period, the absolute cell counts did not change. Intracellular CTLA-4 expression in CD4+ CD25lo/− T cells significantly increased after ipilimumab infusion. We conclude that CTLA-4 blockade by a single infusion of ipilimumab increased CD4+ and CD4+HLA-DR+ T lymphocyte counts and intracellular CTLA-4 expression at the highest dose level. There was no significant change in Treg cell numbers after ipilimumab infusion. These data show that significant changes in T cell populations occur upon exposure to a single dose of ipilimumab. Further studies with multiple doses are needed to explore this phenomenon further and to correlate changes in lymphocyte subpopulations with clinical events.

ABSTRACT

Blast cells from patients with acute myeloid leukemia (AML) commonly express CD64, the high-affinity receptor for immunoglobulin G (FcgammaRI). An immunotoxin (MDX-44) was constructed by coupling humanized anti-CD64 monoclonal antibody (mAb) H22 via a bivalent linker to deglycosylated ricin A-chain (RA). Human leukemia cell lines were incubated with MDX-44 or H22/free RA. The effect of MDX-44 on the proliferation of leukemia cells was assessed by [(3)H]thymidine incorporation. In the presence of interferon-gamma (IFN-gamma), MDX-44 significantly inhibited the proliferation of CD64(+) HL-60, NB4, and U937 cells in 72-h cultures in a dose-dependent manner. The mechanism of action appeared to be the induction of apoptosis, as measured by propidium iodide staining and flow cytometry analysis. However, CD64(-) KG-1a and Daudi cells were not affected by MDX-44/IFN-gamma. Incubating HL-60 cells with MDX-44/IFN-gamma resulted in a 99% decrease in colony-forming units, whereas colony-forming cells in normal bone marrow were not significantly suppressed by such treatment. Cells from 60% of AML patients (6/10) were inhibited by MDX-44/IFN-gamma, and the inhibition was correlated with CD64 expression on these cells (r = 0.65). In a human AML model in NOD/SCID mice, MDX-44/IFN-gamma inhibited 95-98% of peritoneal exudate AML cell proliferation and 85-90% of solid leukemia masses. The effect of MDX-44 on AML cells was dependent on activation of cells by IFN-gamma. MDX-44/IFN-gamma may have value in the therapy of AML cells expressing cell-surface CD64.

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