The objective of this study was to enhance the immunostimulatory efficacy of RNA-transfected DC vaccines by selectively eliminating CD4+
Tregs in metastatic RCC patients. For Treg depletion, we used the recombinant fusion protein denileukin diftitox (DAB389
) in a human vaccination setting. We show that human CD4+
Tregs can be eliminated using a single dose of DAB389
IL-2 without apparent bystander toxicity and without having an impact on the function of other cells expressing CD25. However, DAB389
IL-2 also abrogated DC-mediated activation of T cells in vitro, suggesting that the applicability of this reagent should be restricted to a prevaccination setting (Figure C). These preclinical results provided important information regarding the design of our clinical study, in which DAB389
IL-2 was administered to RCC patients 4 days prior to DC-based vaccination. This time interval was chosen since, unlike antibodies, DAB389
IL-2 is characterized by a short duration of action, with a half-life of approximately 60 minutes, thereby minimizing the possibility of interfering with the ongoing vaccine-induced T cell response.
In this study, DAB389IL-2 profoundly reduced the number of Tregs present in the peripheral blood of RCC patients, reduced levels of peripheral blood–derived FoxP3 transcripts, and abrogated Treg-mediated immunosuppressive activity in vivo. Moreover, significantly higher frequencies of tumor-specific CD8+ T cells could be measured in patients treated with combined DAB389IL-2 and vaccination when compared with subjects receiving the vaccine alone. Also, there was a trend toward an improved CD4+ T cell response after combined therapy. Cumulatively, these data provide several independent lines of evidence that Tregs were depleted in the peripheral blood of RCC patients by using a single dose of the fusion protein DAB389IL-2.
The T cell frequencies achieved after Treg depletion and 3 vaccination cycles were remarkably high with up to 0.90% of all CD8+
T cells demonstrating tumor specificity. No clear correlation between the efficacy of Treg depletion and the magnitude of the vaccine-induced T cell response was observed. Also, serum diphtheria titers did not appear to have an impact on vaccine efficacy (11
). The present study further suggests that the degree of Treg depletion achieved using a single dose of 18 μg/kg may be quite variable and that Treg depletion was transient, with most cells returning after 2 months. However, it should be pointed out that the exact enumeration of Tregs in a vaccination setting is complicated by the fact that CD4+
T cells with negative or intermediate expression levels of CD25 may upregulate expression of CD25 in response to antigenic stimulation, thereby biasing results towards increased detection of Tregs. Our preclinical studies also suggest that the Treg strategy may be geared toward the improvement of T cell responses against relatively weak self antigens such as hTERT or MART-1 antigens but not against immunodominant peptide-derived antigens (Figure D). Accordingly, other studies have recently shown that Tregs effectively suppress the physiologic activation of autoreactive T cells associated with low strength of the antigenic signal while T cells activated with high antigenic signal strength were refractory to this mechanism of suppression (25
). Although in this study, the concept of Treg elimination has been employed in context with RNA-transfected DC-based vaccination, this strategy could potentially be applied to many immune-based approaches of active and passive immunotherapy as well as to classical adjuvants. The information gained from this study will serve as a baseline for further clinical investigation to better define the full potential of this strategy in ultimately achieving antitumor immunity with clinical impact. For such studies it will be critical to collect precise information on Treg depletion and vaccine-induced T cell response and, ultimately, address the clinical efficacy of such strategy in cancer patients.