Ii-Key/HER-2/neu(776-790) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/neu+ tumors

Abstract

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-790) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-gamma-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-443) CTL epitope, as illustrated by either IFN-gamma ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-443) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-790) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-790) hybrid peptides.

Fig. 1

Stimulation of normal donor PBMC with hybrid peptides enhances recognition of native peptide presented onto autologous DC. Mean stimulation index ± SD from three normal donors is shown. NP native peptide, B-F hybrid peptides, HIV Ii-Key/HIV gag(164-76) control hybrid peptide

Fig. 2

Levels of responses to autologous tumor cells after priming with the hybrid peptides. a Graphs were assigned as percent of no, low, intermediate and high responders based on the level of responses [i.e. number of IFN-γ specific spots generated against the autologous tumor cells, induced to express HLA-DR molecules upon IFN-γ treatment, after subtracting the control stimulations with non-IFN-γ treated autologous tumor cells]. The levels of responses are given as fold increase compared to those induced upon priming with the NP. The cut-off for each group was defined randomly. b Recognition of IFN-γ treated tumor cells by autologous CD4+ T cells primed with NP or hybrid peptides B–F. Each dot represents number of IFN-γ specific spots produced by a single patient tested. Bars represent mean values. P values compared to NP: peptide-B = 0,0003, -C = 0,0658, -D = 0,0007, -E = 0,3982, -F = 0,0002. P values for hybrid peptides -C and -E were non-significant compared to NP

Fig. 3

Stimulation of isolated CD4+ T cells with Ii-Key/HER-2/neu hybrid peptides enhances recognition of native HER-2/neu peptide presented onto autologous DCs in a breast cancer patient (a). In (b) similarly stimulated CD4+ cells from two patients with ovarian (pat. ova) and breast (pat. bre) cancer, showed enhanced recognition of their autologous tumor cells. Mean values of IFN-γ specific spots ± SD from quadruplicate wells are shown. Priming CD4+ T cells with the irrelevant control Ii-Key/HIVgag(164-76) hybrid peptide and testing against DCs pulsed with native HER-2/neu(776-90) epitope in (a) or against the autologous tumors in (b), induced a low number of IFN-γ responder cells. NP native peptide; B-F hybrid peptides, HIV irrelevant control peptide

Fig. 4

CD8+ T cells (from a lung cancer patient), primed with autologous CD4+ T cells specific for Ii-Key hybrid peptides, [CD4(B), CD4(D) and CD4(F) induced increased frequencies of HER-2/neu(435-43)-specific CTL as compared to those CD8+ T cells primed with the native peptide, [CD4(NP)], in an IFN-γ ELISPOT assay. HER-2/neu(435-43)-CTL which were either not preincubated with CD4+ T cells [CD8(435)] or were preincubated with non-stimulated CD4+ T cells [CD8(435) + CD4] or with CD4+ T cells primed with the control Ii-key/HIVgag(164-76) peptide [CD8(435) + CD4(HIV)], elicited comparable IFN-γ responses. Mean values ± SD from quadruplicate wells are illustrated

Fig. 5

Increased cytotoxicity of HER-2/neu(435-43)-specific CD8+ T cells, [CD8(435)], from a lung (A) (the same donor as in Fig.4) and ovarian (B) cancer patient upon incubation with autologous CD4+ T cells stimulated with NP or hybrid peptides. Mean values from quadruplicate cultures are shown. The SD was always less than 15% of the means and thus omitted

Fig. 6

Therapeutic efficacy of HER-2/neu(435-43) CTL transferred along with peptide-specific autologous CD4+ T-cells in SCID mice xenografted with the HER-2/neu+ , HLA-A2.1+ human breast tumor cell line SKOV3.A2. CTL and CD4+ T cells (1 × 10⁶ each subset) were ip. injected per mouse in groups of ten SCID mice, which were previously (12-6 days) inoculated sc. on the back with 5 × 10⁵ SKOV3.A2 cells. Tumor growth was caliper-measured and recorded every 4 days in all groups until tumor area had reached a diameter > 200 mm². Data are reported as the mean tumor area of 10 mice per group. The SD was always less than 25% of the means and thus, it was omitted. CTL primed with an irrelevant HLA-A2.1-restricted peptide [gp(154-62)] along with the CD4(B) T cells did not eradicate the growth of transplanted SKOV3. A2 tumor cells similarly as the untreated controls