TriMix and tumor antigen mRNA electroporated dendritic cell vaccination plus ipilimumab: link between T-cell activation and clinical responses in advanced melanoma

Abstract

Background: We previously reported that dendritic cell-based mRNA vaccination plus ipilimumab (TriMixDC-MEL IPI) results in an encouraging rate of tumor responses in patients with pretreated advanced melanoma. Here, we report the TriMixDC-MEL IPI-induced T-cell responses detected in the peripheral blood.

Methods: Monocyte-derived dendritic cells electroporated with mRNA encoding CD70, CD40 ligand, and constitutively active TLR4 (TriMix) as well as the tumor-associated antigens tyrosinase, gp100, MAGE-A3, or MAGE-C2 were administered together with IPI for four cycles. For 18/39 patients, an additional vaccine was administered before the first IPI administration. We evaluated tumor-associated antigen specific T-cell responses in previously collected peripheral blood mononuclear cells, available from 15 patients.

Results: Vaccine-induced enzyme-linked immunospot assay responses detected after in vitro T-cell stimulation were shown in 12/15 patients. Immune responses detected in patients with a complete or partial response were significantly stronger and broader, and exhibited a higher degree of multifunctionality compared with responses in patients with stable or progressive disease. CD8+ T-cell responses from patients with an ongoing clinical response, either elicited by TriMixDC-MEL IPI or on subsequent pembrolizumab treatment, exhibited the highest degree of multifunctionality.

Conclusions: TriMixDC-MEL IPI treatment results in robust CD8+ T-cell responses in a meaningful portion of stage III or IV melanoma patients, and obviously in patients with a clinical response. The levels of polyfunctional and multiantigen T-cell responses measured in patients with a complete response, particularly in patients evidently cured after 5+ years of follow-up, may provide a benchmark for the level of immune stimulation needed to achieve a durable clinical remission.

Trial registration number: NCT01302496.

Keywords: T-lymphocytes; dendritic cells; immunotherapy; melanoma; vaccination.

Conflict of interest statement

Competing interests: The use of dendritic cells electroporated with tumor antigen mRNA and TriMix is topic of a patent (W2009/034172) on which KT is filed as inventor. This patent has been licensed to eTheRNA immunotherapies. BDK and SC are respectively current and former employees of eTheRNA immunotherapies. BN has received financial compensation from Bristol-Myers Squibb for public speaking and participation in advisory board meetings.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

TriMixDC-MEL IPI treatment schedule TriMixDC-MEL vaccines were administered both intradermally (4 million cells) and intravenously (20 million cells) together with IPI every 3 weeks for a total of four administrations. For 18/39 patients, a first vaccine was performed 2 weeks before the first IPI administration. This first administration was omitted for the remaining 21 patients. Patients with SD, PR, or CR at week 24 were eligible to enter an IPI maintenance phase every 12 weeks. IPI was given at 10 mg/kg. CR, complete response; IPI, ipilimumab; PR, partial response; SD, stable disease; TriMixDC-MEL, dendritic cell -based mRNA vaccination plus ipilimumab.

Figure 2

Durable clinical responses on TriMixDC-MEL IPI treatment Kaplan-Meier estimates of PFS and OS. IPI, ipilimumab; OS, overall survival; PFS, progression-free survival; TriMixDC-MEL, dendritic cell -based mRNA vaccination plus ipilimumab.

Figure 3

TriMixDC-MEL IPI treatment results in strong vaccine-specific T-cell responses. (A) Bars show the numbers of patients for whom an IVS ELISPOT response was detected PreVac (white bars) or PostVac (black bars). (B and C) TriMixDC-MEL IPI treatment mediated increase of TAA-specific IVS ELISPOT (B) and IVS ICS (C) response height. Each dot represents the PreVac and PostVac response measured for one patient for one TAA. T-cell responses that were considered as vaccine-specific immune responses are indicated in color (green: tyrosinase; orange: gp100; blue: MAGE-A3; purple: MAGE-C2). The line indicates an equal PreVac and PostVac response. (D) Example of a tyrosinase-specific response as measured with IVS ELISPOT and IVS ICS. (E) Detection of multifunctional CD8+ T-cell responses on TriMixDC-MEL IPI treatment. Each dot represents an IVS ICS response that was considered to be vaccine-specific. The percentage of CD8+ T cells producing either one, two, or three cytokines is shown on the graphs (triangles: PreVac, dots: PostVac). A paired Wilcoxon test was performed to determine statistical significance. (F) Breadth of vaccine responses elicited by TriMixDC-MEL IPI in patients showing an immune response against all vaccine TAA. The bar graphs show the number of enriched clonotypes detected per antigen at the PreVac (white bars) and PostVac (black bars) time point. Clonotypes were considered to be enriched if their frequency in a TAA-stimulated well was >200 fold of their frequency in a Gag (negative control)-stimulated well. The range (min–max) of the enrichment as compared with the Gag-stimulated well is shown above the bars. ICS, intracellular cytokine staining; IVS, in vitro T-cell stimulation; IPI, ipilimumab; ELISPOT, enzyme-linked immunospot assay; SFU, spot forming units; TAA, tumor-associated antigen; TNF, tumor necrosis factor; TriMixDC-MEL, dendritic cell -based mRNA vaccination plus ipilimumab.

Figure 4

TriMix-DC-MEL IPI treatment results in higher numbers of peripheral blood CD62Lhigh Tregs. (A) Percentage of Tregs within CD4+ T-cell population of peripheral blood. Each data point represents the Treg frequency in one patient. Data were analyzed by a Wilcoxon matched-pairs signed rank test. (B) Left: percentage of CD45RA+, HLA-DR+, CD27+, CCR7+, CD62L+, and ICOS+ cells within Treg population of peripheral blood. Each data point shows the PreVac (triangles) or PostVac (dots) result. Results were analyzed with a two-way ANOVA with Sidak’s multiple comparison test. Right: example of CD62L expression profile of Tregs from PreVac (black histogram) and PostVac (red histogram) samples from patient 141. The filled grey histogram shows the negative control not stained with anti-CD62L. ANOVA, analysis of variance; IPI, ipilimumab; TriMixDC-MEL, dendritic cell -based mRNA vaccination plus ipilimumab.

Figure 5

Correlation between immune responses and clinical responses towards TriMixDC-MEL IPI treatment. (A) Each symbol represents an IVS ELISPOT response measured PreVac (triangles) or PostVac (dots) for one patient for one TAA. The bars indicate the mean. A two-way ANOVA with Sidak’s multiple comparisons test was performed to determine statistical significance. (B) The graph indicates the number of TAA for which a vaccine-specific immune response was detected by IVS ELISPOT. Each dot represents one patient. (C) Multifunctional profile of vaccine-specific IVS ICS responses. Each bar shows the percentage of CD8+ T cells producing either one, two or three of the tested cytokines in a TAA-specific fashion on vaccination. (D) The graph shows the percentage of the total CD8+ T-cell IVS ICS response characterized by a multifunctional profile (≥2 cytokines). Each dot represents one vaccine-specific IVS ICS response. The bars indicate the median. A Mann-Whitney test was performed to determine statistical significance. (E and F) The percentages of the total CD8+ T-cell IVS ICS response characterized by a multifunctional profile (E: at least two cytokines, F: three cytokines). Each dot represents one vaccine-specific IVS ICS response. A linear regression was performed to analyze the data. Panels A–F: Red symbols indicate immune responses of patients that showed a CR to the TriMixDC-MEL IPI treatment which is still ongoing after >314 weeks. Blue symbols indicate immune responses of patients having experienced PD after, respectively, 6.77 and 20.45 months but that afterwards obtained CR to pembrolizumab treatment, which is currently still ongoing. Orange symbols indicate patients with a mixed response (complete regression of some of their metastases while others progressed). ANOVA, analysis of variance; CR, complete response; ICS, intracellular cytokine staining; IPI, ipilimumab; IVS, in vitro T-cell stimulation; ELISPOT, enzyme-linked immunospot assay; OS, overall survival; PD, progressive disease; PR, partial response; SFU, spot forming units; SD, stable disease; TAA, tumor-associated antigen; TriMixDC-MEL, dendritic cell -based mRNA vaccination plus ipilimumab.