HER2 Transgenic Mice Research Articles

Genetic Regulation of the Response to Her-2 DNA Vaccination in Human Her-2 Transgenic Mice

Genetic regulation of immune reactivity to Her-2 vaccination and the consequent antitumor effect was tested in human Her-2 transgenic (Tg) mice of C57BL/6 (B6), BALB/c (BALB), and (B6x BALB) F1 (F1) background. Mice were electrovaccinated with Her-2 DNA with or without pretreatment with CD25 monoclonal antibody to remove CD25(hi) regulatory T cells. When CD25(+) T cells were intact, BALB Her-2 Tg mice were more responsive than the other two strains in both humoral and cellular immunities, and their tumor growth was significantly delayed. B6 Her-2 Tg mice responded poorly and F1 mice showed modest immune reactivity, but tumor growth did not change in either strain. Depletion of CD25(hi) T cells before vaccination significantly improved protection from tumor challenge in F1 Her-2 Tg mice. This was associated with elevated levels of Her-2 IgG1, IgG2a, and IgG2c antibodies, and some mice also showed IFN-gamma producing T-cell response. The same treatment induced modest improvement in B6 Her-2 Tg mice. In BALB Her-2 Tg mice, however, depletion of CD25(hi) T cells did not further improve antitumor efficacy. Although their Her-2-specific IgG1 and interleukin-5-secreting T cells increased, the levels of IgG2a and IFN-gamma-secreting T cells did not change. These results are the first to show genetic regulation of the response to a cancer vaccine and an unequal effect of removing CD25(hi) T cells on antitumor immunity. These results warrant individualized treatment plans for patients with heterogeneous genetic backgrounds and possibly differential intrinsic immune reactivity to tumor antigens.

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

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.

The “A, B and C” of Her-2 DNA vaccine development

The development of Her-2 DNA vaccine has progressed through three phases that can be categorized as phase "A": the pursuit of Her-2 as a tumor-associated "antigen", phase "B": tilting the "balance" between tumor immunity and autoimmunity and phase "C": the on-going "clinical trials". In phase "A", a panel of human ErbB-2 or Her-2 plasmids were constructed to encode non-transforming Her-2 derivatives. The immunogenicity and anti-tumor activity of Her-2 DNA vaccines were tested in human Her-2 transgenic mice with or without the depletion of regulatory T cells (Tregs). However, Treg depletion or other immune modulating regimens may increase the risk of autoimmunity. In phase "B", the balance between tumor immunity and autoimmunity was assessed by monitoring the development of experimental autoimmune thyroiditis (EAT). To test the efficacy of Her-2 DNA vaccines in cancer patients, clinical trials have been initiated in phase "C". Significant anti-Her-2 and anti-tumor activity was observed when Her-2 transgenic mice were electro-vaccinated after Treg depletion. Susceptibility to EAT was also enhanced by Treg depletion and there was mutual amplification between Her-2 immunity and EAT development. Although Tregs regulate both EAT and Her-2 immunity, their effector mechanisms may differ. It may be possible to amplify tumor immunity with improved strategies that can by-pass undue autoimmunity. Critical information will be revealed in the next decade to expedite the development of cancer vaccines.

Systemic Spread Is an Early Step in Breast Cancer

It is widely accepted that metastasis is a late event in cancer progression. Here, however, we show that tumor cells can disseminate systemically from earliest epithelial alterations in HER-2 and PyMT transgenic mice and from ductal carcinoma in situ in women. Wild-type mice transplanted with single premalignant HER-2 transgenic glands displayed disseminated tumor cells and micrometastasis in bone marrow and lungs. The number of disseminated cancer cells and their karyotypic abnormalities were similar for small and large tumors in patients and mouse models. When activated by bone marrow transplantation into wild-type recipients, 80 early-disseminated cancer cells sufficed to induce lethal carcinosis. Therefore, release from dormancy of early-disseminated cancer cells may frequently account for metachronous metastasis.

An immunotherapy approach with dendritic cells genetically modified to express the tumor-associated antigen, HER2.

Dendritic cells (DC), genetically modified to express ovalbumin by the retroviral vector GCDNsap, can elicit stronger anti-tumor immunity than those loaded with the peptides. To assess the clinical feasibility of the strategy, such DC were prepared by differentiation of hematopoietic progenitor cells transduced with the human epidermal growth factor receptor 2 (HER2). When inoculated in mice, the DC primed both HER2-specific cytotoxic T lymphocytes and type 1 T helper lymphocytes, resulting in production of HER2-specific antibody. Of importance is that the antibody mediated antibody-dependent cellular cytotoxicity and opsonization. The potent anti-tumor effects were also confirmed by results of experiments using HER2-transgenic mice. Inoculation of HER2-transduced DC resulted in longer disease-free survival of treated mice that showed significant reduction of primary and metastatic tumors. Interestingly, footpad inoculation resulted in stronger anti-tumor effects compared to subcutaneous administration and induced higher levels of the HER2-specific antibody, suggesting that an important role of humoral immunity in anti-tumor effects for malignancies with membrane-type tumor-associated antigens (TAA). Taken together, vaccination of the TAA-transduced DC may represent a promising form of therapy for breast cancers expressing HER2.

Activity of DNA vaccines encoding self or heterologous Her-2/neu in Her-2 or neu transgenic mice

To assess the efficacy of self versus heterologous ErbB-2 vaccines, the reactivity to human and rat ErbB-2 (Her-2 and neu, respectively) DNA vaccines were tested in normal, Her-2 or neu transgenic mice. When immunized with either Her-2 or neu DNA, normal BALB/c and C57BL/6 mice produced cross-reactive T cells, but only antigen specific antibodies. In Her-2 Tg mice, weak to no anti-Her-2 response was induced by either self Her-2 or heterologous neu DNA, demonstrating profound tolerance to Her-2 and the inability to induce anti-Her-2 immunity with either vaccine. In NeuT mice, vaccination with self neu but not heterologous Her-2 DNA induced anti-neu antibodies and delayed spontaneous tumorigenesis. Both neu and Her-2 DNA induced anti-neu T cell response, but depletion of CD8 T cells did not change the delay in tumorigenesis. Therefore, in NeuT mice, both self and heterologous DNA activated anti-neu T cells, although T cell response did not reach sufficient level to suppress spontaneous tumorigenesis. Rather, induction of anti-neu antibodies by self neu DNA is associated with the delay in spontaneous tumor growth. Overall, NeuT mice were more responsive to DNA vaccination than Her-2 Tg mice and this may be associated with the continuous production of neu by the 10 mammary glands undergoing tumor progression.

Early Role of CD4+ Th1 Cells and Antibodies in HER-2 Adenovirus Vaccine Protection against Autochthonous Mammary Carcinomas

HER-2 is an oncogenic tumor-associated Ag that is overexpressed in several human tumors including breast and ovarian cancer. The efficacy and mechanism of a HER-2-expressing recombinant adenoviral vaccine to protect against tumorigenesis was examined using HER-2 transgenic (BALB-neuT) mice, which develop spontaneous breast tumors in all 10 mammary glands, and also using a transplantable mouse tumor model. Vaccination beginning at 6-8 wk of age (through 19 wk of age) prevented development of spontaneous mammary tumors even after 50 wk, whereas the animals in the control groups had tumors in all mammary glands by 25 wk. Such long-term protection after the last boost has not been achieved previously in this transgenic mouse in which the oncogene is continuously spawning tumorigenesis. Using beta(2)-microglobulin-knockout, IFN-gamma-knockout, and B cell-deficient mice, CD4(+) and CD8(+) cell depletion, and Ab transfer studies, we show that induction of anti-HER-2/neu Abs are both necessary and sufficient for protection, and the IgG2a isotype is most effective. In contrast, CD8(+) T cells are not necessary at all, and CD4(+) T cells are necessary for only 36-48 h after immunization to provide help for B cells but not as effector cells. Equal protection in immunized mice deficient in FcgammaRI/III excluded an FcR-mediated mechanism. Anti-HER-2 serum not only inhibited growth of mammary tumor cell lines expressing HER-2 in vitro but also protected mice from tumors in vivo, suggesting a direct action of Ab on the tumor cells. Such a vaccine may provide Ab-mediated protection against HER-2-expressing breast cancers in humans.

Cure of Mammary Carcinomas in Her-2 Transgenic Mice through Sequential Stimulation of Innate (Neoadjuvant Interleukin-12) and Adaptive (DNA Vaccine Electroporation) Immunity

Whereas neoadjuvant therapy is emerging as a treatment option in early primary breast cancer, no data are available on the use of antiangiogenic and immunomodulatory agents in a neoadjuvant setting. In a model of Her-2 spontaneous mammary cancer, we investigated the efficacy of neoadjuvant interleukin 12 (IL-12) followed by "immune-surgery" of the residual tumor. Female BALB/c mice transgenic for the rat Her-2 oncogene inexorably develop invasive carcinomas in all their mammary glands by the 23rd week of age. Mice with multifocal in situ carcinomas received four weekly i.p. injections of 100 ng IL-12 followed by a 3-week rest. This course was given four times. A few mice additionally received DNA plasmids encoding portions of the Her-2 receptor electroporated through transcutaneous electric pulses. The protection elicited by IL-12 in combination with two DNA vaccine electroporations kept 63% of mice tumor-free. Complete protection of all 1-year-old mice was achieved when IL-12-treated mice received four vaccine electroporations. Pathologic findings, in vitro tests, and the results from immunization of both IFN-gamma and immunoglobulin gene knockout transgenic mice and of adoptive transfer experiments all show that IL-12 augments the B- and T-cell response elicited by vaccination and slightly decreases the number of regulatory T cells. In addition, IL-12 strongly inhibits tumor angiogenesis. In Her-2 transgenic mice, IL-12 impairs tumor progression and triggers innate immunity so markedly that DNA vaccination becomes effective at late points in time when it is ineffective on its own.

261 Multi-epitope peptide vaccine and co-administration of IL-12 prevents tumor growth in Her-2 transgenic mice

261 Multi-epitope peptide vaccine and co-administration of IL-12 prevents tumor growth in Her-2 transgenic mice

Human ErbB-2 (Her-2) transgenic mice: a model system for testing Her-2 based vaccines.

Her-2 transgenic (Tg) mice were generated with wild-type human c-ErbB-2 (Her-2) under the whey acidic protein promoter. They are tolerant to Her-2 and appropriate for testing Her-2 vaccines. The expression of transmembrane ErbB-2 from the whey acidic protein-Her-2 cassette and its up-regulation by insulin and hydrocortisone was verified by in vitro transfection. The transgene cassette was microinjected into fertilized eggs from B6C3 (C3H x C57BL/6) females mated with B6C3 males. Transgene-positive mice were backcrossed onto C57BL/6 mice. Human ErbB-2 was expressed in the secretory mammary epithelia during pregnancy and lactation and expressed constitutively in the Bergman glia cells within the molecular layer of the cerebellum. Overt, neoplastic transformation was not detected in any tissue examined. Tolerance to Her-2 was demonstrated by inoculating mice with a syngenic tumor expressing high levels of human ErbB-2. Tumors grew exclusively in Her-2 Tg mice without inducing an Ab response, while the nontransgenic littermates remained tumor free for 10 mo and mounted a robust anti-ErbB-2 Ab response. When immunized five times with plasmid DNA encoding secErbB-2 and GM-CSF, respectively, approximately 33% of the Her-2 Tg mice rejected a lethal challenge of EL-4/E2 tumor cells, whereas all immunized littermates rejected the tumor. Therefore, Her-2 Tg mice express human ErbB-2 in the brain and mammary gland and demonstrated tolerance to ErbB-2 which was partially overcome by DNA vaccination. The breakable tolerance of Her-2 Tg mice resembles that in human and these mice are particularly suited for testing human ErbB-2 based vaccines.

HER-2 DNA and protein vaccines containing potent Th cell epitopes induce distinct protective and therapeutic antitumor responses in HER-2 transgenic mice.

Overexpression of the growth factor receptor HER-2 (c-erbB-2, neu) has transforming potential and occurs in approximately 20-30% of breast and ovarian cancers. HER-2 is a self Ag, but Abs and T cells specific for HER-2 have been isolated from cancer patients, suggesting HER-2 may be a good target for active immunotherapy. We constructed rat HER-2 DNA and protein vaccines containing potent Th cell epitopes derived from tetanus toxin and studied their potency in two strains of mice transgenic for the rat HER-2 molecule. Vaccination with HER-2 DNA protected nontransgenic mice from tumor challenge, but induced only moderate protection in one of the tumor models. However, vaccination with the modified HER-2 protein resulted in almost complete protection from tumor challenge in both tumor models. This protection could be mediated by Abs alone. In addition, protein vaccination efficiently eliminated pre-established tumors in both models, even when vaccination occurred 9 days after tumor implantation. These data demonstrate the potential of HER-2-based vaccines as therapeutic agents for the treatment of cancers overexpressing HER-2.

Distinct responses to modified HER-2 vaccines in two different HER-2 transgenic mouse models lessons for the clinic

Distinct responses to modified HER-2 vaccines in two different HER-2 transgenic mouse models lessons for the clinic

Vaccination with modified HER-2 molecules containing potent helper T cell epitopes induces protective immune responses in HER-2 transgenic mice

Vaccination with modified HER-2 molecules containing potent helper T cell epitopes induces protective immune responses in HER-2 transgenic mice