Antitumor cytotoxic T-cell response induced by a survivin peptide mimic

Abstract

Survivin is a tumor-associated antigen with significant potential as a cancer vaccine target. We have identified a survivin peptide mimic containing human MHC class I epitopes and a potential class II ligand that induces a potent antitumor response in C57BL/6 mice with GL261 cerebral gliomas. This peptide is able to elicit both CD8+ CTL and T helper cell responses in C57BL/6 mice. The corresponding region of the human survivin molecule represented by peptide SVN53-67 is 100% homologous to the murine protein, but SVN53-67 is weakly immunogenic in man. We evaluated several amino acid substitutions in putative human MHC I anchor positions in SVN53-67 to identify potential peptide mimics that could provide an enhanced antitumor immune response against human glioma and primary central nervous system lymphoma (PCNSL) cells in culture. We evaluated survivin peptides with predicted binding to human HLA-A*0201 antigen using peptide-loaded dendritic cells from PBMC of patients with these malignancies. One alteration (M57) led to binding to HLA-A*0201 with significantly higher affinity. We compared the ability of autologous dendritic cells loaded with SVN53-67 peptide and SVN53-67/M57 in CTL assays against allomatched and autologous, survivin-expressing, human malignant glioma and PCNSL cells. Both SVN53-67 and SVN53-67/M57 produced CTL-mediated killing of malignant target cells; however, SVN53-67/M57 was significantly more effective than SVN53-67. Thus, SVN53-67/M57 may act as a peptide mimic to induce an enhanced antitumor CTL response in tumor patients. The use of SVN53-67/M57 as a cancer vaccine might have application for cancer vaccine therapy.

Figures

Fig. 1

a RT–PCR of human glioma specimens for survivin expression. Frozen sections from intracranial GL261 tumors were analyzed for survivin expression by direct immunofluorescence. b Isotype control mAb; c anti-survivin mAb on glioma showing high survivin expression levels (Images were captured at 400× magnification)

Fig. 2

Survivin peptide mimic vaccine amino acid sequence and subsequently derived MHC class I binding epitopes. Additionally, the SVN53-67/M57 15-mer is predicted to bind the following MHC class II alleles: HLA-DRB1*0301 (DR17), HLA-DRB1*0401 (DR4Dw4), HLA-DRB1*0701 and HLA-DRB1*1501 (DR2b)

Fig. 3

a MHC class I peptide competitive displacement assays. HLA-A*0201 binding of survivin peptides based upon the core SVN56-64 peptide. IC50 represents concentration of survivin peptide required to displace HPV 18-27 (known MHC I ligand) pre-loaded on human T2 cells. Control peptides (Flu and gp100) are used as known immunogenic MHC class I ligands. b Pentamer binding assay of SVN56-64 wild type and SVN56-64/M57 mimic peptides showing differential binding affinity of each peptide for MHC class I

Fig. 4

a Survival of C57BL6 mice with intracerebral GL261 glioma implants. Mice received intracerebral implants of 1 × 105 GL261 cells, followed by vaccination beginning 4 days after tumor implantation to simulate a therapeutic setting. Vaccinations were repeated every 7 days up to a total of 3 immunizations. b High field strength MRI of glioma-implanted C57BL/6 mice without (left) and with (right) peptide mimic vaccination. c CTL responses against GL261 glioma cells obtained in vitro using the splenocytes of mice vaccinated with SVN53-67/M57. d Intracellular IFN-γ production in vitro of CD4+ cells from mice in response to SVN53-67/M57 immunization. Mouse cytokine intracellular control cells (ebiosciences) were used as positive controls for IFNγ expression

Fig. 5

Ex vivo cytotoxic T lymphocyte (CTL) responses against allogeneic and autologous human glioma, PCNSL and CLL cells to assess specificity and efficacy of cell killing toward tumor cells displaying MHC class I contextualized survivin peptides. a HLA-A* 0301 patient effector cells versus allogeneic-mismatched HLA-A* 0201 human U87 target glioma cells; b HLA-A*0201 patient effector cells versus allogeneic-matched U87 glioma cells; c HLA-A* 0301 patient effector cells versus allogeneic-matched patient-derived tumor cells; d HLA-A*0301/HLA-A*2901 patient effector cells versus autologous patient-derived glioma cells. e HLA-A*2901/ HLA-A*3002 patient effector cells versus autologous patient-derived PCNSL target cells; f HLA-A*01/HLA-A*24 patient effector cells versus autologous patient-derived CLL target cells. Legend: Control DC = Flu/58-66; Positive control = gp100/209-217; SVN wt = SVN53-67; SVN Mimic = SVN53-67/M57

Fig. 6

IFNγ accumulation in culture medium as measured by ELISA. PBMC were stimulated by indicated peptide-loaded DC in culture. Medium was analyzed on days 2, 10 and 14 and results are displayed as pg/ml detected in culture medium. Control DC are non-specific control stimulated DC and T-cell control represents wells of non-stimulated T cells used to assess background. Results are mean values of triplicate wells ± SEM. The figure shown is representative of three patient experiments

Fig. 7

Pentamer binding from patient-derived CTL generation showing that SVN53-67/M57 peptide mimic can produce CD8+ T cells that are reactive to wild-type peptide. a HLA-A*0201 pentamer binding CD8+ T cells specific for SVN56-64/M57 peptide mimic as a result of indicated antigen stimulation. Percentage shown is relative to all cells analyzed from restimulated cultures. b Corresponding CTL assay data for patient cells used in (a)