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Clinical Trial
. 2007 Oct 1;179(7):4919-28.
doi: 10.4049/jimmunol.179.7.4919.

Administration of a CD25-directed immunotoxin, LMB-2, to patients with metastatic melanoma induces a selective partial reduction in regulatory T cells in vivo

Daniel J Powell Jr  1 , Aloisio Felipe-Silva, Maria J Merino, Mojgan Ahmadzadeh, Tamika Allen, Catherine Levy, Donald E White, Sharon Mavroukakis, Robert J Kreitman, Steven A Rosenberg, Ira Pastan
Affiliations
Clinical Trial

Administration of a CD25-directed immunotoxin, LMB-2, to patients with metastatic melanoma induces a selective partial reduction in regulatory T cells in vivo

Daniel J Powell Jr et al. J Immunol. .

Abstract

CD25+ CD4+ T regulatory (Treg) cells regulate peripheral self tolerance and possess the ability to suppress antitumor responses, which may in part explain the poor clinical response of cancer patients undergoing active immunization protocols. We have previously shown that in vitro incubation of human PBMC with LMB-2, a CD25-directed immunotoxin, significantly reduced CD25+ FOXP3+ CD4+ Treg cells without impairing the function of the remaining lymphocytes. In the current study, eight patients with metastatic melanoma were treated with LMB-2 followed by MART-1 and gp100-specific peptide vaccination. LMB-2 administration resulted in a preferential, transient reduction in mean circulating CD25+ CD4+ T cell number, from 83 +/- 16 cells/microl to a nadir of 17 +/- 5 cells/microl, a 79.1% reduction. FOXP3 analysis revealed a less robust depletion with mean FOXP3+ CD4+ Treg cell number decreasing from 74 +/- 15 cells/microl to 36 +/- 8 cells/microl, a 51.4% reduction. FOXP3+ CD4+ Treg cells that survived LMB-2-mediated cytotoxicity expressed little or no CD25. Similar to the peripheral blood, immunohistochemical analysis showed a 68.9% mean reduction in FOXP3+ CD4+ Treg cell frequency in evaluable lesions. Despite inducing a reduction in Treg cell numbers in vivo, LMB-2 therapy did not augment the immune response to cancer vaccination and no patient experienced an objective response or autoimmunity. These data demonstrate the capacity of a CD25-directed immunotoxin to selectively mediate a transient partial reduction in circulating and tumor-infiltrating Treg cells in vivo, and suggest that more comprehensive Treg cell elimination may be required to bolster antitumor responses in patients with metastatic melanoma.

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Figures

FIGURE 1
FIGURE 1
LMB-2 administration causes a selective, transient elimination of circulating CD25+ Treg cells in vivo. A, A transient reduction in the mean ALC was induced after the first dose of LMB-2 on day 0. Each dot represents the percent change in ALC relative to pretreatment counts for each patient. The boxed line indicates the mean percent change in ALC for all patient values on each day. B, Changes in T cell subset counts following LMB-2 infusion. Each line represents the mean percent change in the number of the indicted T cell subset relative to pretreatment counts for all available patients. CD25 expression was evaluated with the 4E3 Ab clone. C, CD25 and FOXP3 expression by circulating CD4+ T cells is reduced after LMB-2 administration. Representative dot plots show the expression of CD25 (4E3 clone; y-axis) and FOXP3 (x-axis) on CD4+CD3+ gated cells from patient 5 at the indicated time after first infusion. D, CD25 expression by CD4+CD3+ gated T cells was transiently reduced following LMB-2 administration in all patients. The mean level of CD25 expression by CD4 T cells from all patients is shown, as detected using Ab clones 2A3 (which recognizes the same epitope region as LMB-2) or 4E3 (which binds a unique region of CD25). E, The mean level of FOXP3-expressing CD4+CD3+ T cells was reduced for 1 wk after initiation of LMB-2 therapy. F, LMB-2 selectively eliminates FOXP3-expressing CD4+ T cells that express high levels of CD25 in vivo. The frequency CD4+CD3+ -gated cells expressing FOXP3 and high levels of CD25 or intermediate/negative CD25 were assessed over time. The mean frequency of CD25highFOXP3+CD4+ T cells and CD25int/negativeFOXP3+CD4+ T cells is shown. *, Significance (p ≤ 0.05) on the indicated day; ns, changes that were not significant.
FIGURE 2
FIGURE 2
A reduction in the frequency of tumor-infiltrating FOXP3+CD4+ T cells in metastatic melanoma lesions following LMB-2 administration. Immunohistochemical analysis for FOXP3 (brown), CD4 (red, in A and C), and CD8 (red, in B and D) was performed on pretreatment (A and B) or day 8 posttreatment (C and D) metastatic melanoma lesions from patient 1. Arrows denote FOXP3-expressing cells; ×40 magnification was used.
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