Immunologic responses to xenogeneic tyrosinase DNA vaccine administered by electroporation in patients with malignant melanoma

Abstract

Background: Prior studies show that intramuscular injection and particle-mediated epidermal delivery of xenogeneic melanosomal antigens (tyrosinase or Tyr, gp100) induce CD8(+) T cell responses to the syngeneic protein. To further define the optimal vaccination strategy, we conducted a phase I study of in vivo electroporation (EP) of a murine Tyr DNA vaccine (pINGmuTyr) in malignant melanoma patients.

Methods: Human leukocyte antigen (HLA)-A1, A2, A24 or B35 stage IIb-IV melanoma patients received up to five doses of the mouse tyrosinase DNA vaccine by EP every three weeks at dose levels of 0.2 mg, 0.5 mg, or 1.5 mg per injection. Peripheral blood mononuclear cells (PBMC) were collected, cultured with a peptide pool containing eight HLA class I-restricted Tyr-specific T-cell epitopes, and analyzed by HLA-A*0101-restricted tetramers and intracellular cytokine staining (ICS).

Results: Twenty-four patients received ≥1 dose of the pINGmuTyr vaccine; PBMCs from 21 patients who completed all five doses were available for Tyr immune assays. The only common toxicity was grade 1 injection site reaction. Six of 15 patients (40%) in the 1.5 mg dose cohort developed Tyr-reactive CD8(+) T cell responses following stimulation, defined as a ≥3 standard deviation increase in baseline reactivity by tetramer or ICS assays. No Tyr-reactive CD8(+) T cell response was detected in the 0.2 mg and 0.5 mg dose cohort patients. Epitope spreading of CD8(+) T cell response to NY-ESO-1 was observed in one patient with vitiligo. One patient subsequently received ipilimumab and developed an enhanced Tyr-reactive response with polyfunctional cytokine profile. After a median follow-up of 40.9 months, median survival has not been reached.

Conclusions: A regimen of five immunizations with pINGmuTyr administered by EP was found to be safe and resulted in Tyr-reactive immune responses in six of 15 patients at 1.5 mg dose cohort.

Trial registration: ClinicalTrials.gov NCT00471133.

Keywords: DNA vaccine; Electroporation; Epitope spreading; Immune response; Melanoma patient; Tyrosinase.

Figures

Figure 1

Increase in tyrosinase243-251HLA*A010-restricted tetramer-reactive CD8+T cells following mouse tyrosinase DNA vaccination. Multiparameter flow cytometry was performed at baseline, prior to the third vaccination (week 10) and after the fifth vaccination (Week 16). Patient peripheral blood mononuclear cells (PBMCs) were cultured for 10 days with a pool of tyrosinase peptides. Representative dot plots from Patient Tyr-26 reveal an increase in the frequency of tetramer-reactive CD8+ T cells in this patient, with the peak at Week 10.

Figure 2

Changes in tyrosinase146-156and tyrosinase243-251HLA*A0101-restricted tetramer-reactive CD8+T cells following mouse tyrosinase DNA vaccination. Each point refers to the mean of triplicate values. Most patients also underwent two peripheral blood draws at baseline one week apart prior to receiving vaccination. The values at baseline represent the mean of both of these time-points. * Refers to patients with increase in tetramer-reactive CD8+ cells. (Patient Tyr-9 scored positive for both Tyr tetramers, patient Tyr-26, 27 and Tyr-28 had a single Tyr tetramer positive after vaccination).

Figure 3

CCR7, CD45RA, CD27 and CD28 subpopulations in tyrosinase243-251HLA*A0101-restricted tetramer-reactive CD8+T cells are consistent with an effector phenotype. PMBCs were analyzed by tetramer assay after in vitro culture with tyrosinase peptide pool. Representative dot plots for CD3+CD8+ T cells of patient Tyr-26 at Week 16 show the expression of CCR7, CD45RA, CD28 and CD27 on tyrosinase243-251 HLA*A0101 tetramer reactive CD8+ T cells (A). Further characterization in contour plots of CD3+CD8+tetramer+ T cells revealed that these cells were CCR7-CD45RA-CD27loCD28mid, consistent with an effector phenotype (B).

Figure 4

Increase in tyrosinase-reactive CD8+IFN-γ+T cells following mouse tyrosinase DNA vaccination. Intracellular cytokine staining was performed by multiparameter flow cytometry after culturing patient PBMCs with a tyrosinase peptide epitope pool for 10 days. These are representative dot plots of CD3+CD8+ T cells from Patient Tyr-25 who had an increase in CD8+IFN-γ+ cells following vaccination. There was evidence of polyfunctionality, including cells that were both IFN-γ+, and either CD107a+, MIP-1β+ or TNF-α+.

Figure 5

Changes in frequency of tyrosinase-reactive CD8+IFN-γ+T cells following mouse tyrosinase DNA vaccination. Each point refers to the mean of triplicate values in intracellular cytokine staining assays. Most patients also underwent two peripheral blood draws at baseline one week apart prior to receiving vaccination. The values at baseline represent the mean of both of these time-points. * Refers to patients with increase in CD8+ IFN-γ + T cells.

Figure 6

CTLA-4 blockade induced tyrosinase-specific CD8+IFN-γ+T cell responses. ICS was performed on PBMCs from Patient Tyr-25 at various time-points. He developed a tyrosinase-specific CD8+IFN-γ+ T cell response following vaccination. Subsequently, he developed recurrent disease and was treated with ipilimumab. A persistent tyrosinase-specific CD8+IFN-γ+ response was noted prior to ipilimumab therapy, which increased with ipilimumab therapy and peaked after the second dose (at Week 7).

Figure 7

Mouse tyrosinase DNA vaccination and subsequent NY-ESO-1 specific tetramer and IFN-γ response. Patient Tyr-2 was known to have baseline seropositivity for NY-ESO-1. Thawed PBMCs from pre- and post- pINGmuTyr vaccination were cultured for 10 days with NY-ESO-1 overlapping peptides before tetramer analysis and ICS. Following vaccination, there was an increase in (A) HLA/B*35 NY-ESO-194-102 tetramer-reactive CD8+ T cells and (B) NY-ESO-1 specific CD4+ and CD8+ IFN-γ+ T cells.