De Giovanni et al. Breast Cancer Research 2014, 16:R10 http://breast-cancer-research.com/content/16/1/R10 RESEARCH ARTICLE Open Access Vaccines against human HER2 prevent mammary carcinoma in mice transgenic for human HER2 Carla De Giovanni1,2, Giordano Nicoletti3, Elena Quaglino4, Lorena Landuzzi3, Arianna Palladini1, Marianna Lucia Ianzano1, Massimiliano Dall’Ora1, Valentina Grosso1, Dario Ranieri1, Roberta Laranga1, Stefania Croci1, Augusto Amici5, Manuel L Penichet6, Manuela Iezzi7, Federica Cavallo4, Patrizia Nanni1,2* and Pier-Luigi Lollini1,2 Abstract Introduction: The availability of mice transgenic for the human HER2 gene (huHER2) and prone to the development of HER2-driven mammary carcinogenesis (referred to as FVB-huHER2 mice) prompted us to study active immunopreventive strategies targeting the human HER2 molecule in a tolerant host. Methods: FVB-huHER2 mice were vaccinated with either IL-12-adjuvanted human HER2-positive cancer cells or DNA vaccine carrying chimeric human-rat HER2 sequences. Onset and number of mammary tumors were recorded to evaluate vaccine potency. Mice sera were collected and passively transferred to xenograft-bearing mice to assess their antitumor efficacy. Results: Both cell and DNA vaccines significantly delayed tumor onset, leading to about 65% tumor-free mice at 70 weeks, whereas mock-vaccinated FVB-huHER2 controls developed mammary tumors at a median age of 45 weeks. In the DNA vaccinated group, 65% of mice were still tumor-free at about 90 weeks of age. The number of mammary tumors per mouse was also significantly reduced in vaccinated mice. Vaccines broke the immunological tolerance to the huHER2 transgene, inducing both humoral and cytokine responses. The DNA vaccine mainly induced a high and sustained level of anti-huHER2 antibodies, the cell vaccine also elicited interferon (IFN)-γ production. Sera of DNA- vaccinated mice transferred to xenograft-carrying mice significantly inhibited the growth of human HER2-positive cancer cells. Conclusions: Anti-huHER2 antibodies elicited in the tolerant host exert antitumor activity. Introduction successfully targeted for tumor prevention and therapy. The ErbB-2 oncogene (HER2 in humans) is amplified HER2-targeted therapies based on monoclonal anti- and overexpressed in 20% to 30% of aggressive breast bodies (such as trastuzumab and pertuzumab) and on cancers, as well as in fractions of tumors of the ovary, small-molecule inhibitors are in widespread clinical use. stomach and others [1-3]. HER2 codes for a membrane Even though generally well-tolerated, such treatments tyrosine kinase; therefore, it can be targeted by have some drawbacks: cost, need for continuous antibodies and immune effector cells. Its oncogenic treatment, increased risk of cardiac toxicity and, mainly, function, coupled to membrane expression, led to the frequent onset of resistance to therapy. The search for definition of an oncoantigen [4,5], that is, an oncogene anti-HER2 vaccines able to break the patient’s tolerance which is also a tumor-associated antigen that can be toward HER2 is being pursued to elicit effective anti- tumor immune responses and immunological memory [6-8]. Anti-HER2 vaccines could be employed in a * Correspondence: [email protected] 1Laboratory of Immunology and Biology of Metastasis, Department of therapeutic setting, even in combination with other Experimental, Diagnostic and Specialty Medicine, Alma Mater Studiorum therapies, but most likely could be used in an adjuvant University of Bologna, Viale Filopanti 22, I-40126 Bologna, Italy setting to prevent the development of metastases or to 2Interdepartmental Centre for Cancer Research “Giorgio Prodi”, Alma Mater Studiorum University of Bologna, Via Massarenti 9, I-40138 Bologna, Italy Full list of author information is available at the end of the article © 2014 De Giovanni et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. De Giovanni et al. Breast Cancer Research 2014, 16:R10 Page 2 of 11 http://breast-cancer-research.com/content/16/1/R10 target the early stages of the disease, such as in situ car- a primer set specific to human growth hormone exons cinoma [9]. 4 and 5, which are included in the transgene backbone, Preclinical studies on the prevention of mammary car- as reported previously [19]. Mice were inspected weekly cinogenesis driven by the rat HER2 gene (normal or mu- by palpation. Progressively growing masses larger than tated) have shown that several immune approaches can 0.3 cm in diameter were scored as tumors. The mice hamper the neoplastic process, ranging from the admin- were killed when the diameter of one of the tumors istration of cytokines, such as interleukin 12 (IL-12) exceeded 1.7 cm. Nontransgenic FVB/NCrl (FVB) fe- [10], to active vaccination approaches [4,5]. Cell vaccines male mice were purchased from Charles River Labora- have shown high potency only when coupled with strong tories (Calco, Como, Italy). For xenograft experiments, − − − − adjuvant stimuli (such as allogeneic stimulation and IL-12) we used the immunodeficient Rag2 / ;Il2rg / mice [11,12]. Cytokines other than IL-12 were found to be far (kindly provided by Drs Nomura and Ito, Central Insti- less efficient [12]. The adjuvant effect of IL-12 in cell vac- tute for Experimental Models, Kawasaki, Japan) [21]. In cine also was found to be effective in curing minimal re- vivo experiments were performed in compliance with sidual disease [13]. DNA vaccines also have proved to be the Italian and European guidelines and were approved highly effective [14-16]. by the Institutional Review Board of the University of Although passive targeting of the human HER2 (huHER2) Bologna. homologue—for example, with antibodies—can easily be studied in vivo against human HER2-positive can- Cells cers grown as xenografts in immunodeficient mice, ac- The HER2-positive human ovarian carcinoma cell line tive immune approaches require immunocompetent mice SK-OV-3 was cultured in RPMI 1640 medium (Invitro- tolerant to huHER2. Some huHER2 transgenic lines gen, Milan, Italy) supplemented with 10% fetal bovine [17,18] were found not to develop spontaneous mammary serum (FBS) and maintained at 37°C in a humidified tumors and were used only in vaccination challenge ex- atmosphere with 5% CO2. Other human cell lines with periments to study the efficacy of anti-HER2 vaccines. different HER2 expression were used as well: MDA- Tumor-prone mice transgenic for huHER2, obtained by MB-453 (breast cancer origin, medium to high HER2 Finkle and co-workers [19], allow the study of immuno- expression) [21], MCF-7 (breast cancer origin, low prevention of autochthonous tumor onset through vac- HER2 expression) and SJ-RH4 (rhabdomyosarcoma, cines targeting the normal huHER2 molecule. These mice null HER2 expression) [22]. We established a cell line, (referred to as FVB-huHER2 herein) carry a wild-type which we refer to as syn-HER2, from a mammary carcinoma huHER2 gene under the control of the mouse mammary of a FVB-huHER2 mouse. This cell line showed high tumor virus (MMTV) promoter and show spontaneous expression of huHER2 and tumorigenicity in syngeneic development of mammary tumors in most female mice in hosts. the second semester of life. A few data have been reported on the prevention of tumors of these mice by passive Plasmids transfer of antibodies [19] and by an anti-idiotype active Chimeric human/rat HER2 plasmid electroporated vac- immune approach [20]. cine (HuRT), previously described in detail [16], derived In our present study, we used FVB-huHER2 transgenic from pVAX1 (Invitrogen), encodes a chimeric protein in mice to study anti-huHER2 vaccine strategies (a xenogeneic which the first 390 extracellular NH2-terminal residues whole-cell vaccine and a DNA human/rat chimeric vac- are from huHER2 and the remaining extracellular and cine) to break tolerance to huHER2. We had two aims: transmembrane residues from rat HER2/neu. Empty vec- (1) to evaluate vaccine efficacy in the immunopreven- tor pVAX1 was used as an experimental control. Large- tion of huHER2-driven mammary tumors, and (2) to scale production and purification of the plasmids were study whether anti-HER2 antibodies resulting from a performed with EndoFree Plasmid Giga kits (QIAGEN, break of tolerance could inhibit human tumors growing Valencia, CA, USA) as previously reported [15]. as xenografts. Cytokine and vaccine treatments Methods The cell vaccine was formulated as mitomycin C–treated Mice huHER2-overexpressing human cancer cells (SK-OV-3) FVB-huHER2-transgenicmicewereobtainedfromGenen- associated with exogenous administration of recombin- tech (line MMTV.f.hu.HER2#5(Fo5) on FVB background; ant murine IL-12 to provide the specific huHER2 anti- South San Francisco, CA, USA) [19]. They carry the full- gen combined with adjuvants (xenogenicity and IL-12). length, normal huHER2 gene under the control of the The cell vaccination schedule was reported previously. MMTV promoter. FVB-huHER2 mice were bred in our Briefly it was based on a 4-week cycle. During the first animal facilities and genetically screened by PCR using 2 weeks, mice received four twice-weekly intraperitoneal De Giovanni et al. Breast Cancer Research 2014, 16:R10 Page 3 of 11 http://breast-cancer-research.com/content/16/1/R10 (i.p.) vaccinations with 2 × 106 proliferation-blocked Cytokine production (mitomycin C–treated [23]) SK-OV-3 cells in 0.4 ml of Spleen cells were collected from vaccinated and control phosphate-buffered saline (PBS). During the third week, mice after at least three vaccination cycles. Interferon γ mice received daily i.p. administration of murine IL-12 (IFN-γ) production by spleen mononuclear cells was followed by 1 week of rest [11]. The IL-12 dose was evaluated after in vitro culture for 6 days alone (spontan- 50 ng per day in the first vaccination cycle and 100 ng eous release) or in the presence of proliferation-blocked per day in subsequent cycles.