Enhanced and long term immunogenicity of a Her-2/neu multi-epitope vaccine conjugated to the carrier CRM197 in conjunction with the adjuvant Montanide

Abstract

Background: We previously identified three short single peptides (P4, P6 and P7) representing different B-cell epitopes on the extracellular domain of Her-2/neu for a vaccine that was tested in a phase-I clinical trial. Here we describe the improvement of the multi peptide vaccine by fusing the single peptides to a hybrid peptide P467.

Methods: After coupling to either virosomes or to diphtheria toxoid CRM197 (CRM), the hybrid peptide was tested in different concentrations in combination with either Montanide or Aluminium hydroxide (Alum) in preclinical studies.

Results: Already low amount (10 μg) of P467 conjugated to CRM led to faster onset of high antibody levels compared to the P467-virosome. The formulation P467-CRM-Montanide induced higher serum IgG antibody titers, compared with P467-CRM-Alum, as examined by ELISA using recombinant Her-2/neu or Her-2/neu natively expressed on the tumor cell line SK-BR-3. Compared to P467-CRM-Alum, higher in vitro production of IL-2 and IFNγ in the Montanide-immunized mice was induced after re-stimulation of splenocytes with CRM but also with P467, indicating a clear Th1-biased response. In contrast to the single B cell peptides, the hybrid peptide led to T cell proliferation and cytokine production as CD4 T cell epitopes were generated in the fusion region of the single peptides P4 and P6 or P6 and P7. Additionally, a significantly higher proportion IFNγ-producing CD8+ T cells was found in the P467-CRM-Montanide immunized mice, probably by Montanide-driven bystander activation. Importantly, anti-P467 IgG antibodies exhibited anti-tumor properties and the combination of anti-P467 specific IgG with Herceptin® was found to inhibit the proliferation of Her-2/neu-overexpressing cell line SK-BR-3 in a significantly higher capacity than Herceptin® alone.

Conclusions: Fusion of the B cell peptides has led to additional generation of CD4 T cell epitopes, and this P467-multi epitope vaccine was found to induce polyclonal antibody responses with anti-proliferative capacity against Her-2/neu. The hybrid vaccine together with Montanide induced higher and long-lasting antibody levels, Th1-biased cellular responses being superior to vaccination with the single B cell peptides. This vaccine formulation is now planned to be evaluated in a phase Ib/II study in Her-2/neu overexpressing cancer patients.

Keywords: Adjuvant; Her-2/neu; Humoral and cellular response; Hybrid peptide; Mice; Th1-deriving response.

Fig. 1

Experimental design. Female Balb/C mice were immunized with different amounts of P467 and P647 hybrid peptides, conjugated to either CRM or virosomes (1a; Experiment-I), or with different amounts of P467-CRM administered together with Alum or Montanide (1b; Experiment-II). For examining the kinetic of the immune responses, additional blood samples were taken in two months intervals for the period of 6 months after the last immunization. The occasions for immunization (black arrows) and bleeding (grey arrows) are indicated

Fig. 2

Level of serum IgG antibody responses against recombinant extracellular Her-2/neu. a Kinetic of the antibody responses induced after 2, 3, and 4 immunizations with 30 μg of the hybrid peptide P467 conjugated to virosomes or CRM, are shown. b Comparison of the levels of the antibody responses induced after 3 immunizations with virosomal constructs expressing a mixture of single peptides (10 μg or 25 μg), virosomal constructs expressing the P467 peptide (15 μg or 30 μg), or the CRM-conjugated P467 administered together with Montanide (10 μg or 25 μg) is shown. The OD values are based on ELISA experiments as described in materials and methods. The statistical analysis was performed using ANOVA (two-way) and linear contrasts. Significant differences are denoted by asterisks

Fig. 3

Levels of serum IgG, IgG1 and IgG2a antibody responses against P467 and recombinant extracellular Her-2/neu. The levels of serum IgG, IgG1 and IgG2a against P467 (3a, 3b and 3c) or recombinant extracellular Her-2/neu (3d, 3e and 3f), induced after 3 immunizations with different concentrations of the conjugate administered with Alum or Montanide, are shown. The OD values are based on ELISA experiments as described in materials and methods. The statistical analysis was performed using ANOVA (two-way) and linear contrasts. Significant differences are denoted by asterisks. ns, not significant

Fig. 4

Kinetic of serum IgG antibody responses against P467. The levels of serum IgG antibody responses, induced 2, 4 and 6 months after the last immunization with different amounts of P467-CRM conjugate administered with Alum or Montanide, are shown. * The mice in this group were sacrificed before the six-month period, and their splenocytes were used for the T cell epitope mapping analysis (Table 2). The OD values are after subtraction of the background (i.e. pre-immunized mice), and based on ELISA experiments as described in materials and methods. The statistical analysis was performed using Generalized Estimation Equations. Significant difference is denoted by ***

Fig. 5

Levels of cytokines, IL-2, IFNγ and IL-5, produced in vitro. Levels of IL-2, IFNγ and IL-5 produced in vitro in splenocytes of mice immunized with different amounts of P467-CRM conjugate administered with Alum or Montanide, after re-stimulation with CRM (5a, 5b and 5c) or P467 (5d, 5e and 5f), are shown. Splenocytes of the immunized mice were used and re-stimulated, as described in materials and methods. The statistical analysis was performed using ANOVA (two-way) and linear contrasts. The measured levels of the controls (Alum Montanide and CRM) in Fig. 5d and f were lower than the background, and therefore were set to zero. Significant differences are denoted by asterisks. ns, not significant

Fig. 6

T cell stimulation capacity of the hybrid peptide P467 and single B cell epitope peptides. Splenocytes of mice immunized with 25 μg of P467-CRM together Montanide were re-stimulated with the hybrid peptide P467 or the single B cell epitope peptides P4, P6 and P7, as described in materials and methods. The bars indicate stimulation indexes (SI) in the presence of each examined peptide. The horizontal line indicates the cut-off of 2.0, above which the calculated values of SI were considered as significant

Fig. 7

Level of INFγ-producing CD8+ T cells. The proportions of INFγ − producing CD8+ T cells in mice immunized with 25 μg of P467-CRM conjugate administered with Alum or Montanide, are shown. The statistical analysis was performed using t test. Significant difference is denoted by asterisks

Fig. 8

Proliferation inhibition of SK-BR-3 cells in vitro. [3H]-thymidine proliferation assay demonstrating the effects of purified anti-P467 rabbit IgG antibodies on the Her-2/neu-overexpressing human breast cancer cell line SK-BR-3 and the human melanoma cell line 518.A2 as control cells (Her-2/neu negative). Data are expressed in percentage of inhibition, based on the mean (+ SEM) of the tested antibody isolates; cpm values of untreated wells were set to 100%. Significant difference is denoted by *. ns, not significant