Single dose denileukin diftitox does not enhance vaccine-induced T cell responses or effectively deplete Tregs in advanced melanoma: immune monitoring and clinical results of a randomized phase II trial

Abstract

Background: Depletion of CD25(+) Tregs improves anti-tumor immunity in preclinical models. Denileukin diftitox is a recombinant fusion protein of human IL-2 and diptheria toxin fragment that also can kill CD25(+) T cells. Prior clinical trials of denileukin diftitox suggested reduction of FoxP3(+) Tregs and some clinical responses.

Method: To investigate the immunologic effects of denileukin difitox on vaccine-specific immune responses in melanoma, a randomized clinical trial of single dose denileukin diftitox prior to vaccination versus vaccination alone in subjects with HLA-A2(+) metastatic melanoma was performed. Treatment included randomization to a 4-peptide vaccine (Melan-A, gp100, MAGE3 and NA17 with GM-CSF emulsified in Montanide) alone or after single dose of denileukin diftitox (18 mcg/kg). Vaccine was given every 2 weeks for 3 doses and, absent clinical progression, continued every 2 weeks. Blood and tumor biopsies were obtained pretreatment and after 3 vaccinations for immunologic assessments.

Results: In 17 treated subjects there were no drug-related G3-4 adverse events. One partial response and 8 stable disease were observed in 9 subjects (4 DD: 5 vaccine only) with no impact of denileukin diftitox on time to progression. Total peripheral Tregs were not significantly altered, and in 1 patient biopsy intra-tumoral FoxP3 transcripts were not reduced following denileukin diftitox. ELISA for IL2R-α demonstrated no impact on outcomes by soluble CD25 level. Immune monitoring suggested the development of modest vaccine-specific CD8(+) T cell responses in the control group, however immunization efficacy was actually reduced in the denileukin diftitox group.

Conclusion: Our results indicate that denileukin diftitox did not effectively deplete Tregs, augment T cell responses, or improve clinical activity in melanoma. Clinicaltrials.gov ID: NCT00515528; Registered August 9, 2007.

Keywords: Denileukin difitox; Immunotherapy; Melanoma; Tregs; Vaccine.

Figures

Fig. 1

Representative flow cytometric gating for CD4+CD25+FoxP3+ cells. Peripheral blood mononuclear cells were isolated using density gradient centrifugation before and after patient received denileukin diftitox treatment versus no treatment. Cells were labeled with anti-CD4, anti-CD25, and anti-FoxP3 Abs from the Treg staining kit (Miltenyi Biotech, Inc.) and then analyzed using a MACSQuant analyzer

Fig. 2

CD4+CD25hiFoxP3+ Treg in peripheral blood before and after denileukin diftitox treatment. Peripheral blood mononuclear cells were isolated using density gradient centrifugation before and after patient received denileukin diftitox treatment versus no treatment. In the denileukin difitox arm, peripheral blood mononuclear cells were isolated at the time of consent (pre) and before the first vaccination (post). Tregs were first labeled with anti-CD4, anti-CD25, and anti-FoxP3 Abs from the Treg staining kit (Miltenyi Biotech, Inc.) and then analyzed using a MACSQuant analyzer. Paired student t-test was used to determine whether there was a statistical difference between the pre- and post-treatment samples in each cohort

Fig. 3

Intratumoral FoxP3 mRNA transcripts before and after treatment with and without denileukin diftitox. Fresh biopsies was collected before and after treatment when feasible and snap frozen in liquid nitrogen. RNA samples were isolated using the Trizol regent, and cDNA was synthesized using the High Fidelity cDNA Synthesis Kit (Roche Applied Science). qRT-PCR was then performed to analyze the FoxP3 transcripts levels on ABI-7300 qPCR instrument (Applied Biosystem). β-actin were used as an internal control

Fig. 4

Soluble interleukin-2 receptor-α in patients treated or untreated with denileukin diftitox. Blood was drawn before and after subjects were treated on the trial. Serum was aliquoted and stored at -80 °C until analysis. Soluble interleukin-2 receptor (soluble CD25) levels were analyzed by ELISA. Student t-test (assuming different variance in each group) was used to determine whether there was a statistical difference between the pre and post samples in each cohort and between the two cohorts

Fig. 5

Circulating antigen-specific CD8+ T cells in patients treated or untreated with denileukin diftitox. Peripheral blood mononuclear cells were seeded on anti-IFN-γ Ab-coated ELISPOT plates. 5 μM of peptide was then added to each well and incubated overnight. The plates were then incubated with the biotin-labeled anti-IFN-γ Ab and developed according to manufacturer’s procedure. The plates were read and analyzed on CTL-ImmunoSpot S6 Core Analyzer from Cellular Technology Ltd. Each point represents the numerical difference of Ag-specific CD8+ T cells in PBMCs post-vaccine and pre-treatment for each patient. Each point represents an individual patient. Paired student t-test was used to determine whether there was a difference between the pre- and post- samples in each cohort