Immunotherapy with MVA-BN®-HER2 induces HER-2-specific Th1 immunity and alters the intratumoral balance of effector and regulatory T cells

Stefanie J. Mandl 0 1 Ryan B. Rountree 0 1 Katie Dalpozzo 0 1 Lisa Do 0 1 John R. Lombardo 0 1 Peter L. Schoonmaker 0 1 Ulrike Dirmeier 0 1 Robin Steigerwald 0 1 Thierry Giffon 0 1 Reiner Laus 0 1 Alain Delcayre 0 1 0 U. Dirmeier R. Steigerwald Bavarian Nordic GmbH, Frauenhoferstrasse 13, 82152 Martinsried, Germany 1 S. J. Mandl (&) R. B. Rountree K. Dalpozzo L. Do J. R. Lombardo P. L. Schoonmaker T. Giffon R. Laus A . Delcayre Department of Research , BN ImmunoTherapeutics, 2425 Garcia Ave, Mountain View, CA 94043, USA MVA-BN -HER2 is a new candidate immunotherapy designed for the treatment of HER-2-positive breast cancer. Here, we demonstrate that a single treatment with MVA-BN -HER2 exerts potent anti-tumor efficacy in a murine model of experimental pulmonary metastasis. This anti-tumor efficacy occurred despite a strong tumormediated immunosuppressive environment characterized by a high frequency of regulatory T cells (Treg) in the lungs of tumor-bearing mice. Immunogenicity studies showed that treatment with MVA-BN -HER2 induced strongly Th1-dominated HER-2-specific antibody and T-cell responses. MVA-BN -HER2-induced anti-tumor activity was characterized by an increased infiltration of lungs with highly activated, HER-2-specific, CD8?CD11c? T cells accompanied by a decrease in the frequency of Treg cells in the lung, resulting in a significantly increased ratio of effector T cells to Treg cells. In contrast, administration of HER2 protein formulated in Complete Freund's Adjuvant (CFA) induced a strongly Th2-biased immune response to HER-2. However, this did not lead to significant infiltration Stefanie J. Mandl and Ryan B. Rountree contributed equally to this work. - MVA-BN -HER2 is currently being developed as a candidate for breast cancer immunotherapy and has shown biological activity in ongoing Phase I clinical trials (NCT0048277). MVA-BN -HER2 is a Modified Vaccinia Ankara-based recombinant vaccine vector derived from MVA-BN (IMVAMUNE ), a highly attenuated clonal virus currently in development as a smallpox vaccine [1]. MVA-BN -HER2 encodes a modified form of human epidermal growth factor receptor 2 (HER-2), referred to as HER2. HER2 comprises of the extracellular domains of HER-2 and was further modified to encode two promiscuous T helper cell epitopes from tetanus toxin. This modification has been shown to improve its immunogenicity in a tolerant environment [2]. Successful cancer immunotherapy will most likely require the induction of comprehensive innate and Th1 adaptive immune responses that provide effector mechanisms able to directly kill tumor cells while combating the inflammatory/immunosuppressive responses induced by the growing tumor. In this regard, MVA-BN has been shown to confer protection in animal models of smallpox through the induction of strong Th1 adaptive as well as innate immune responses [37]. These intrinsic immune properties are beneficial features to mediate adequate immune responses to MVA-BN -encoded transgenes expressing tumor targets. This was demonstrated herein, by comparing the induction of HER-2-specific immune responses mediated by either MVA-BN -HER2 or HER2 formulated in Freunds adjuvant. When tested in a murine model of experimental pulmonary metastasis (CT26HER-2), MVA-BN -HER2-mediated immune responses translated into potent anti-tumor efficacy, while HER2 formulated in Freunds adjuvant had only modest effects. Multiple cell types can exert immunosuppressive functions within tumors, thereby hampering the development of successful immunotherapies [812]. Among these, regulatory T cells (Treg) appear to play a critical role in the progression of a number of cancers including breast cancer [1317]. Even if tumor-specific effector cells are initially recruited into the tumor, the recruitment of Treg cells into the same site can result in an unproductive anti-tumor response, and this is believed to play a major part in the failure of many cancer immunotherapies presently in development [14, 18]. Furthermore, recent research in mouse and human showed that, regardless of the overall number of Treg cells in the tumor, the local balance of Treg to effector T cells (Teff) is critical to the success of immunotherapy and ultimately survival [1922]. The murine CT26 colon carcinoma cell line has been shown to be a potent inducer of immunosuppressive mechanisms [2325] making it a good model to evaluate the interplay between anti-tumor immunity and tumor-induced immunosuppression. When injected intravenously (i.v.), CT26 cells give rise to experimental pulmonary metastasis while mediating the infiltration of tumor-bearing lungs by a large number of IL-10-secreting Treg [25]. Data described here demonstrate that MVA-BN -HER2 treatment induced Th1-dominated HER-2-specific cellular and humoral immunity, reduced the amount of Treg cells in the tumor and altered the intratumoral balance of effector and regulatory T cells resulting in potent anti-tumor efficacy against CT26-HER-2 tumors. Our findings suggest that MVABN -HER2 generated an anti-tumor response in line with the current paradigm of an immune signature [26] that could lead to protective immunity in humans and warrants further testing of MVA-BN -HER2 in clinical trials. Materials and methods Animals, tumor cell lines, MVA-BN -vectors and HER2 proteins Female BALB/c (H-2d) mice were obtained from Harlan SpragueDawley, Inc. CT26-HER-2 is a cell line derived from an undifferentiated murine colon adenocarcinoma that was transduced with human HER-2/neu [27]. CT26HER-2 cells were grown in IMDM ? 10% bovine calf serum (Cellgro, Mediatech, VA). MVA-BN and MVABN -HER2 were produced for BNIT by Bavarian Nordic (BN; Martinsried, Germany) [28]. The HER2 transgene encodes the extracellular domains of HER-2 and a portion of the trans-membrane domain but lacks the intracellular domains. Furthermore, it encodes the T helper cell epitopes p2 and p30 from tetanus toxin [2]. The HER2 protein used in combination with CFA is similar to the protein expressed by the MVA-BN -transgene with the exception that the trans-membrane domain (9 AA) is absent and the protein has a C-terminal His tag. Analysis of immune responses Serum antibody titers and the frequency of IFN-c-producing T cells were determined by standard ELISA and ELISPOT, respectively. Functionality of the T cells was tested by in vivo CTL assay and intracellular IFN-c stain (for details, see Online Resource ESM_1_Materials and Methods MOESM1: Supplemental material 1.). 5E5 CT26-HER-2 cells were injected i.v. into female BALB/c mice. On day four, mice were treated intraperitoneally (i.p.) with 10 lg HER2 in CFA or 5E7 TCID50 of MVA-BN or MVA-BN -HER2 unless indicated otherwise in the Figure legend. Fourteen days after tumor implantation, mice were euthanized, bled by cardiac puncture, and lungs and spleens were collected. For assessment of tumor burden, lungs were washed in PBS, tapped dry on a tissue and weighed. The tumor burden was (...truncated)