Her-2 DNA versus cell vaccine: immunogenicity and anti-tumor activity

Abstract

Direct comparison and ranking of vaccine formulations in pre-clinical studies will expedite the identification of cancer vaccines for clinical trials. Two human ErbB-2 (Her-2) vaccines, naked DNA and whole cell vaccine, were tested side-by-side in wild type and Her-2 transgenic mice. Both vaccines can induce humoral and cellular immunity to the entire repertoire of Her-2 epitopes. Mice were electro-vaccinated i.m. with a mixture of pGM-CSF and pE2TM, the latter encodes Her-2 extracellular and transmembrane domains. Alternatively, mice were injected i.p. with human ovarian cancer SKOV3 cells that have amplified Her-2. In wild type mice, comparable levels of Her-2 antibodies (Ab) were induced by these two vaccines. However, T cell immunity and protection against Her-2(+) tumors were superior in DNA vaccinated mice. In BALB Her-2 transgenic (Tg) mice, which were tolerant to Her-2, DNA and cell vaccines were administered after regulatory T cells (Treg) were removed by anti-CD25 mAb. Again, comparable levels of Her-2 Ab were induced, but DNA vaccines rendered greater anti-tumor activity. In B6xDR3 Her-2 Tg mice that expressed the autoimmune prone HLA-DR3 allele, higher levels of Her-2 Ab were induced by SKOV3 cell than by Her-2 DNA. But anti-tumor activity was still more profound in DNA vaccinated mice. Therefore, Her-2 DNA vaccine induced greater anti-tumor immunity than cell vaccine, whether mice were tolerant to Her-2 or susceptible to autoimmunity. Through such side-by-side comparisons in appropriate pre-clinical test systems, the more effective vaccine formulations will emerge as candidates for clinical trials.

Fig. 1

Induction of anti-Her-2 immunity by DNA versus cell vaccine in wild type mice. C57BL/6 (a, c) or BALB/c (b, d) mice were immunized twice, 2 weeks apart, by i.m. electro-vaccination with pE2TM and pGM-CSF or by i.p. injection with 2 × 10⁶ SKOV3 cells. Control mice received blank vector pCMV. The inset shows Her-2 expression on SKOV3 cells measured by anti-Her-2 mAb TA-1 (shaded histogram). Normal mouse IgG control is shown in open histogram. a, b Anti-Her-2 IgG levels in individual mice were measured by flow cytometry 2 weeks after second vaccination as described in “Materials and methods”. c, d PBL were collected 2 weeks after second vaccination and cells from each group were pooled. Her-2-specific T cells were enumerated by IFN-γ ELISPOT essay as described in “Materials and methods”

Fig. 2

Protection against tumor growth by DNA versus cell vaccines in wild type mice. C57BL/6 or BALB/c mice were immunized with pE2TM and pGM-CSF or with SKOV3 cells twice, as described in Fig. 1 and mice were challenged s.c. with 2 × 10⁵ Her-2-transfected tumor cells at 2 weeks after the last vaccination. Tumor growth is expressed as % tumor-free mice. The number of mice per group is indicated on the graphs. a Immunized C57BL/6 mice were challenged with EL4/E2 tumors, b Immunized BALB/c mice were challenged with D2F2/E2 tumors. c SKOV3 immunized BALB/c mice were challenged with D2F2/E2 tumors. Some of the immunized mice were depleted of CD8⁺ T cells using mAb 2.43. * P < 0.05, ** P < 0.005 compared to vector–control mice by log-rank test

Fig. 3

Cross-protection against neu-expressing tumors by pE2TM or SKOV3 vaccination. BALB/c mice were immunized with pE2TM or SKOV3 as described. a Anti-neu IgG levels in individual mice were measured 2 weeks after second vaccination by flow cytometry. b Immune splenocytes were collected at the same time. Neu-reactive T cells were enumerated by IFN-γ ELISPOT essay after in vitro stimulation with 3T3/NKB cells. Mice were inoculated s.c. with 2 × 10⁵ D2F2/neu (c) or TUBO (d) tumor cells. There were five to ten mice per group. *P < 0.05, ** P < 0.001 compared to vector-immunized

Fig. 4

Induction of anti-Her-2 immunity by DNA versus cell vaccines in BALB Her-2 Tg mice. a BALB Her-2 Tg mice were injected i.p. with 0.5 mg anti-CD25 mAb, PC61, 10 days before mice were immunized by i.m. electro-vaccination with blank vector, pE2TM and pGM-CSF or by i.p. injection with 2 × 10⁶ SKOV3 cells. The vaccination was repeated three times every 2 weeks. b Her-2 Ab was measured by flow cytometry at 2 weeks after each vaccination. c PBL were collected 2 weeks after the last vaccination and cells from each group were pooled. Her-2-specific T cells were enumerated by IFN-γ ELISPOT essay as described in “Materials and methods”. d At 2 weeks after the last immunization, mice were challenged s.c. with 2 × 10⁵ D2F2/E2 tumor cells. ** P < 0.005 compared to vector-immunized group

Fig. 5

Induction of anti-Her-2 immunity by DNA versus cell vaccines in B6xDR3 Her-2 Tg mice. B6xDR3 Her-2 Tg mice were depleted of CD4⁺CD25^hi Tregs and immunized four times, as in Fig. 4. a Her-2 Ab levels were measured at 2 weeks after the last vaccination. b IgG1 and IgG2c Her-2 Ab induced by DNA and cell vaccines were measured by flow cytometry as described in “Materials and methods”. c At 2 weeks after the last vaccination, mice were inoculated s.c. with 2 × 10⁵ EO771/E2 tumor cells. * P < 0.05 compared to vector or SKOV3 immunized groups