Combining human and rat sequences in her-2 DNA vaccines blunts immune tolerance and drives antitumor immunity

Abstract

Immune tolerance to tumor-associated self-antigens poses a major challenge in the ability to mount an effective cancer vaccine response. To overcome immune tolerance to HER-2, we formulated DNA vaccines that express both human HER-2 and heterologous rat Neu sequences in separate plasmids or as single hybrid constructs that encode HER-2/Neu fusion proteins. Candidate vaccines were tested in Her-2 transgenic (Tg) mice of BALB/c (BALB), BALB/cxC57BL/6 F1 (F1), or C57BL/6 (B6) background, which exhibit decreasing immune responsiveness to HER-2. Analysis of various cocktails or hybrid vaccines defined a requirement for particular combination of HER/2/Neu sequences to effectively prime immune effector cells in HER-2 Tg mice. In B6 HER-2 Tg mice, rejection of HER-2-positive tumors protected mice from HER-2-negative tumors, providing evidence of epitope spreading. Our findings show that a strategy of combining heterologous antigen with self-antigens could produce a potent DNA vaccine that may be applicable to other tumor-associated antigens.

Figure 1

A, Her-2 DNA vaccine constructs. B, cocktail vaccine in BALB Her-2 Tg mice. Mice were electrovaccinated four times, every 2 wk with 50 µg of either pE2_™ or pNeu_™, or a cocktail of both at half the dosage. pGM-CSF (50 µg) was included in each vaccine formulation. Control mice received pVax1 and pGM-CSF. Sera were collected after each vaccination and Her-2–specific antibodies of individual mice were measured by flow cytometry. C, Her-2–responsive IFN-γ–secreting T cells in pooled PBL were measured by ELISPOT assay after the second and fourth vaccinations. PBL were incubated with 3T3/EKB for 48 h before the assay and 3T3/KB cells were used as controls. D, at 2 wk following the fourth vaccination, all mice were challenged s.c. with 2 × 10⁵ D2F2/E2, and tumors were palpated weekly. There were 7 to 10 mice per group (*, P < 0.05).

Figure 2

Hybrid vaccines in BALB Her-2 Tg mice. A, mice were vaccinated four times with pE2_™, pE2-Neu_™, or pNeu-E2_™. Her-2 antibodies in individual mice were measured after each vaccination. IgG₁ and IgG_2a levels were analyzed after the last vaccination. B, Her-2–reactive IFN-γ–secreting cells in pooled PBL were measured after the second and fourth vaccination. C, at 2 wk following the last vaccination, mice were challenged with 2 × 10⁵ D2F2/E2 and tumor growth was monitored by weekly palpation. There were 4 to 10 mice per group (*, P < 0.001).

Figure 3

Hybrid vaccines in (BALB × B6) F1 Her-2 Tg mice. A, mice were electrovaccinated four times with pE2_™, pE2-Neu_™, or pE2-Neu_500™ with or without Treg depletion by CD25 mAb. B, Her-2 antibodies in individual mice were measured after each vaccination. C, IFN-γ–secreting T cells in pooled PBL were measured after four vaccinations. D, in a repeat experiment, vaccinated mice were challenged with 2 × 10⁵ D2F2/E2 or E0771/E2 s.c. and tumor growth was monitored weekly. There were six to seven mice per group (*, P < 0.01).

Figure 4

Hybrid vaccines in B6 Her-2 Tg mice. A, B6 Her-2 Tg mice were depleted of Treg 1 wk prior to the first vaccination. Her-2 antibodies of individual mice were measured after each vaccination. B, Her-2–reactive T cells in pooled PBL were monitored by ELISPOT following the last vaccination and after 48 h of stimulation with TC-1/ E2 cells; TC-1 cells were used as controls. C, at 2 wk after the final vaccination, mice were challenged with 2 × 10⁵ E0771/E2. Tumors were monitored by weekly palpation. There were five to eight mice per group. Tumor-free mice were re-challenged with the parental tumor line, E0771 (*, P < 0.01).

Figure 5

The efficacy of one preventive and three therapeutic vaccines. A, BALB Her-2 Tg mice received 2 × 10⁵ D2F2/E2 tumors 11 d after a single protective vaccination. Mice then received three weekly vaccinations, starting 3 d after tumor inoculation. B, Her-2 antibodies were measured after each vaccination and tumor growth was monitored weekly. There were six to eight mice per group. C, expression of Her-2 by primary cultures of D2F2/E2 tumor was measured by mAb TA-1. D, B6 Her-2 Tg mice were inoculated with 2 × 10⁵ E0771/E2 tumor cells following the same vaccination scheme, except that CD25 mAb was given 1 wk before the first vaccination. Inset, Her-2 expression on E0771/E2 tumor outgrowth in pVax1 group. There were 9 to 10 mice per group (*, P < 0.05; **, P < 0.01).