The results presented herein demonstrate that the unarmed IL-2Rα-specific antibody daclizumab effectively eliminates TRegs
in TMZ treated patients with GBM without decreasing effector T-cell populations or impairing vaccine-stimulated immunity. In vitro
, these antibodies enhance IFN-γ production to an antigen-specific and nonspecific stimulus in CD4+
T-cells, and while IFN-γ secretion by CD8+
T-cells was blunted, this was rescued with the addition of IL-15, a homeostatic cytokine that would be present in patients recovering from chemotherapy-induced lymphopenia. This supports our hypothesis that IL-2Rα-specific antibodies may have differential effects on TRegs
and anti-tumor effector T-cells in the milieu of homeostatic cytokines which would be seen in patients treated with lymphodepleting chemotherapies, such as TMZ, which is now standard-of-care for patients with GBM. Enrollment on this study was halted due to unanticipated discontinuation of the availability of daclizumab by the manufacturer. However, when assessed in our randomized saline-controlled pilot study “ZAP-IT”, daclizumab administration was safe, depleted TRegs
, did not deplete CD4+
effector T-cells and increased the ratio of CD4+
effectors to TRegs
. Importantly, decreased TReg
numbers strongly correlated with heightened vaccine-induced humoral responses, suggesting TReg
depletion may augment vaccine-induced humoral immunity. Finally, unlike other approaches to enhancing immune response in cancer patients 
, we saw no evidence of toxicity despite the dramatic reduction in regulatory T-cell numbers that we observed.
depletion strategies, such as the IL-2 targeted toxins denileukin diftitox and LMB-2, have been used in clinical studies and have had partial success in the in vivo
reduction of regulatory T-cells 
. However, these strategies have limitations not found when using unarmed MAb blockade. Denileukin diftitox targets the IL-2 moiety itself allowing indiscriminate targeting of the lower affinity IL-2ßγ receptors which are expressed on a broader subset of cells including memory T-cells. Thus, denileukin diftitox cannot distinguish between TRegs
and activated and memory T-cells expressing any of the IL-2 receptors and may even stimulate TRegs
. Alternative strategies that employ IL-2Rα-targeted immunotoxins, such as LMB-2, still allow indiscriminate killing of all IL-2Rα-expressing cells including recently activated, vaccine-induced effector T-cells that express IL-2Rα.
We and others have recently shown, however, that unarmed anti-IL-2Rα antibodies may function differently and not have a direct cytotoxic effect 
. Rather, these antibodies may impair regulatory T-cells by blocking IL-2 receptor signaling through their cognate receptor. TRegs
are known to be uniquely dependent on the high affinity IL-2 receptor for their function and survival 
and MAbs that block IL-2Rα have been shown to significantly reduce regulatory T-cell activity in preclinical models 
. While these antibodies would also bind IL-2Rα on activated T-cells, activated effectors may not require this signaling, as others have demonstrated that IL-2 signals during priming are required for robust secondary memory T-cell responses and that activated T-cells are not dependent on IL-2 signaling during the primary response 
. It is additionally quite conceivable that by the time patients are diagnosed and treated, that most tumor antigens actually represent memory T-cells which would not be dependent on IL-2 signaling to generate secondary immune responses. Furthermore, homeostatic cytokines such as IL-7 and IL-15, that are prevalent during lymphopenia, have been shown to be able to substitute for IL-2 signaling in activated effector cells 
. Thus, when anti-IL-2Rα MAbs are employed in the unique host environment that exists after therapeutic TMZ-induced lymphodepletion, vaccine-stimulated anti-tumor T-cells may be independent of IL-2 signaling whereas TRegs
will remain dependent. This differential effect should lead to increased effector
ratios as we have seen here, but remains wholly dependent on a lymphodepleted environment. Given the prior difficulties in eliminating TRegs
without impairing T-cell effectors, it has been controversial whether or not the depletion of TRegs
would enhance immune responses. Our data demonstrating the inverse correlation between TReg
frequency and vaccine-stimulated antibody levels suggests that reducing TRegs
may improve vaccine-induced immunity and warrants further investigation. Of note, TReg
depletion using daclizumab was incomplete in these patients using a single intravenous administration, suggesting either incomplete saturation of IL-2Rα receptors at the dose used (1 mg/kg), downregulation or shedding of IL-2Rα from the surface of TRegs
that renders some cells refractory to an antibody dependent elimination, or a refractory population of FOXP3+ CD4+ TRegs
that is not amenable to elimination by anti-IL-2Rα MAbs treatment. We did not differentiate in this study, for instance, whether thymic-derived natural TRegs
) versus peripherally converted TRegs
are preferentially depleted by anti-IL-2Rα blockade. This is of importance since, recent studies have demonstrated that thymic-derived TRegs
predominate in patients with malignant brain tumors 
. The early and significant depletion of TRegs
shortly after antibody administration in patients after surgical resection, suggests that n TRegs
are likely effectively depleted by this treatment but determination of the effects of anti-IL-2Rα MAbs on TReg
subsets constitute an important area for future research.
The results of our trial examining the impact of daclizumab and anti-tumor vaccination differ widely from the recent work of Jacobs et al.
in which the administration of daclizumab successfully depleted TRegs
but impaired vaccine-stimulated T-cell function and prevented antibody formation. In our study, robust PEPvIII-specific humoral responses were present in both the saline and daclizumab arms and the presence of class-switching indicates that functional CD4+
T-cell help must have been provided. Additionally, our study demonstrates that lower TReg
levels actually correlate with improved anti-PEPvIII antibody responses. Key differences between our study and the work of Jacobs et al.
include type of vaccination (peptide versus DC), randomization (randomized versus not randomized) as well as administration and dose of daclizumab (1 mg/kg at vaccination versus 0.5 mg/kg 4 or 8 days prior to vaccination). However, we believe the fundamental difference is that our application of daclizumab and anti-tumor vaccination occurs in the context of lymphopenia and it is this setting that permits daclizumab to selectively deplete TRegs
while leaving vaccine-stimulated anti-tumor immunity intact.
Although promising, our study does have a number of limitations, one of which is despite being randomized, blinded and placebo-controlled; the number of patients enrolled in this trial is small due to discontinuation of the availability of daclizumab. However, these results have been reproduced in two separate phase I trials we have conducted more recently. In the first trial, patients were vaccinated against Cytomegalovirus
, pp65, a tumor antigen now known to be specifically expressed in GBM 
. While patients treated with vaccine alone have a median progression free survival of only 15.4 months, patients in the second study 
treated with a combination of vaccination and concomitant daclizumab have a progression-free survival of 27.2 months. While these are also small studies, these results may be significant given that the expected progression-free survival is 6.9–8.2 months in this patient population 
. While the EGFRvIII-specific vaccine has been previously shown to stimulate predominantly humoral responses, in these other trials we are additionally examining differences in T-cell responses. The results of our cumulative data provide a safe, novel and much needed method for depleting TRegs
without impairing activated effector T-cells. Unfortunately, the discontinuation of the availability of daclizumab precluded further study of the effects of this drug on immunologic responses. A chimeric monoclonal antibody targeted CD25, basiliximab, however is currently available and studies evaluating the use of this antibody to selectively deplete TRegs
are underway. The utility of repeated administration of anti-IL-2Rα MAbs during recovery from lymphopenia and the effects of dose-escalation of TMZ to achieve greater and sustained lymphopenia constitute potential avenues for exploitation of the use of anti-IL-2Rα MAbs in cancer immunotherapy.