Vaccination with human anti-trastuzumab anti-idiotype scFv reverses HER2 immunological tolerance and induces tumor immunity in MMTV.f.huHER2(Fo5) mice

Abstract

Introduction: Novel adjuvant therapies are needed to prevent metastatic relapses in HER2-expressing breast cancer. Here, we tested whether trastuzumab-selected single-chain Fv (scFv) could be used to develop an anti-idiotype-based vaccine to inhibit growth of HER2-positive tumor cells in vitro and in vivo through induction of long-lasting HER-specific immunity.

Methods: BALB/c mice were immunized with anti-trastuzumab anti-idiotype (anti-Id) scFv (scFv40 and scFv69), which mimic human HER2. Their sera were assessed for the presence of HER2-specific Ab1' antibodies and for their ability to reduce viability of SK-OV-3 cells, a HER2-positive cancer cell line, in nude mice. MMTV.f.huHER2(Fo5) transgenic mice were immunized with scFv40 and scFv69 and, then, growth inhibition of spontaneous HER2-positive mammary tumors, humoral response, antibody isotype as well as splenocyte secretion of IL2 and IFN-γ were evaluated.

Results: Adoptively-transferred sera from BALB/c mice immunized with scFv40 and scFv69 contain anti-HER2 Ab1' antibodies that can efficiently inhibit growth of SK-OV-3 cell tumors in nude mice. Similarly, prophylactic vaccination with anti-Id scFv69 fully protects virgin or primiparous FVB-MMTV.f.huHER2(Fo5) females from developing spontaneous mammary tumors. Moreover, such vaccination elicits an anti-HER2 Ab1' immune response together with a scFv69-specific Th1 response with IL2 and IFN-γ cytokine secretion.

Conclusions: Anti-trastuzumab anti-Id scFv69, used as a therapeutic or prophylactic vaccine, protects mice from developing HER2-positive mammary tumors by inducing both anti-HER2 Ab1' antibody production and an anti-HER2 Th2-dependent immune response. These results suggest that scFv69 could be used as an anti-Id-based vaccine for adjuvant therapy of patients with HER2-positive tumors to reverse immunological tolerance to HER2.

Figure 1

Anti-HER2 antibodies are present in sera from BALB/c mice immunized with anti-Id scFv40 and scFv69. (A) ELISA of pooled sera from mice (n = 20/group) immunized with scFv40, scFv69 or HER2-Fc. Values are presented as mean ± SD of the Absorbance at 490 nm from three independent experiments. Flow cytometry analysis in (B) HER2-overexpressing SK-OV-3 and (C) HER2-negative CHO cells of the HER2-specific response of pooled sera from mice immunized with PBS, anti-Id scFv40, scFv69 or with HER2-Fc. Sera were recovered at Day 42 post-vaccination. Antibody binding was detected using a FITC-labeled goat anti-mouse antibody. FITC-labeled secondary antibody alone was used as a negative control to determine the level of background fluorescence.

Figure 2

Inhibition of cell viability and analysis of SK-OV-3 tumor progression in nude mice. (A) Inhibition of cell viability in SK-OV-3 Luc cells following treatment with sera from BALB/c mice immunized with PBS (NoMS), scFv40 (scFv40MS), scFv69 (scFv69MS) or with HER2-Fc (HER2-FcMS). Cell number was proportional to the emitted luminescence and results were normalized to the fluorescence of untreated cells. Grey boxes represent the 25^th and 75^th percentiles with the medians as black lines; whiskers mark the smallest and largest non-outlier observations, and outliers are indicated by dots. (B) Analysis of tumor progression in nude mice (n = 5/group) xenografted with HER2-overexpressing SK-OV-3-Luc tumor cells and then adoptively transferred by i.p. injection of 200 μl of scFv40MS, scFv69MS, HER2-FcMS or NoMS (negative control), diluted 1:5, at Day 4 after xenograft. Positive controls were animals treated with trastuzumab (200 μg/injection). Tumor growth was evaluated by measuring the emitted luminescence once a week following injection of luciferin. Bioluminescence imaging of nude mice xenografted with SK-OV-3-Luc tumor cells and treated with NoMS (C) or scFv69MS (D) was performed at the end of the treatment (Day 27 after xenograft).

Figure 3

Analysis of the HER2-specific antibody response in sera of MMTV.f.huHER2(Fo5) transgenic mice. Mice were immunized with PBS (n = 4) (A) or anti-Id scFv69 (n = 5) (B). HER2-specific antibody level was measured in sera collected before the first immunization (pre-immune sera) and at the time of the maximum response during the study (Day 42) (immune sera). Each dot represents an immunized mouse.

Figure 4

In vivo prophylactic vaccination with anti-Id scFv protects against spontaneous mammary tumor development in MMTV.f.huHER2(Fo5) transgenic mice. The Kaplan-Meier disease-free survival plots illustrate the age of appearance of mammary tumors in (A) virgin females vaccinated with PBS (negative control), anti-HER2 trastuzumab, scFv69 or HER2-Fc and (B) primiparous females vaccinated with PBS (negative control), anti-Id scFv40 or scFv69, and HER2-Fc.

Figure 5

Analysis of the immune responses induced by vaccination with anti-Id scFv69. IgG1, IgG2a and IgG2b isotype distribution of anti-scFv69 (A) and anti-HER2 (B) antibodies in sera from MMTV.f.huHER2(Fo5) transgenic mice immunized with PBS (upper panels) or scFv69 (lower panels); (C) IgG1/IgG2a ratio of the anti-scFv69 or anti-HER2 response in transgenic mice immunized with PBS or scFv69.