@phdthesis{Huang2013,
  author    = {Huang, Ting},
  title     = {Vaccinia Virus-mediated Therapy of Solid Tumor Xenografts: Intra-tumoral Delivery of Therapeutic Antibodies},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-91327},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Over the past 30 years, much effort and financial support have been invested in the fight against cancer, yet cancer still represents the leading cause of death in the world. Conventional therapies for treatment of cancer are predominantly directed against tumor cells. Recently however, new treatments options have paid more attention to exploiting the advantage of targeting the tumor stroma instead. Vaccinia virus (VACV) has played an important role in human medicine since the 18th century as a vaccination against smallpox. In our laboratory, the recombinant, replication-competent vaccinia virus, GLV-1h68, was shown to enter, colonize and destroy cancer cells both in cell culture, and in vivo, in xenograft models (Zhang, Yu et al. 2007). In addition, combined therapy of GLV-1h68 and anti-VEGF immunotherapy significantly enhanced antitumor therapy in vivo (Frentzen, Yu et al. 2009). In this study, we constructed several new recombinant VACVs carrying genes encoding different antibodies against fibroblast activation protein (FAP) in stroma (GLV-1h282), nanobody against the extracellular domain of epidermal growth factor receptor (EGFR, GLV-1h442) or antibodies targeting both vascular endothelial growth factor (VEGF) and EGFR (GLV-1h444) or targeting both VEGF and FAP (GLV-1h446). The expression of the recombinant proteins was first verified using protein analytical methods, SDS-gel electrophoresis, Western blot analysis, immunoprecipitation (IP) assays and ELISA assays. The proteins were detected after infection of the cells with the different VACVs and the recombinant proteins purified by affinity adsorption. The purified antibodies were shown to specifically bind to their respective antigens. Secondly, the infection and replication capability of all the virus strains was analyzed in cell culture using several human tumor cell lines (A549, FaDu or DU145), revealing that all the new recombinant VACVs were able to infect cancer cells with comparable efficiency to the parental viruses from which they were derived. Thirdly, the antitumor efficacy of the new recombinant VACVs was evaluated in vivo using several human cancer xenograft models in mice. In A549 and DU145 xenografts, the new recombinant VACVs exhibited an enhanced therapeutic efficacy compared to GLV-1h68 with no change in toxicity in mice. In the FaDu xenograft, treatment with GLV-1h282 (anti-FAP) significantly slowed down the speed of tumor growth compared to GLV-1h68. Additionally, treatment with the recombinant VACVs expressed the various antibodies achieved comparable or superior therapeutic effects compared to treatment with a combination of GLV-1h68 and the commercial therapeutic antibodies, Avastin, Erbitux or both. Next, the virus distribution in tumors and organs of treated mice was evaluated. For most of the viruses, the virus titer in tumors was not signficantly diffferent than GLV-1h68. However, for animals treated with GLV-1h282, the virus titer in tumors was significantly higher than with GLV-1h68. This may be the reason for enhanced antitumor efficacy of GLV-1h282 in vivo. Lastly, the underlying mechanisms of therapeutic antibody-enhanced antitumor effects were investigated by immunohistochemistry. Blood vessels density and cell proliferation in tumors were suppressed after treatment with the antibody-encoded VACVs. The results indicated that the suppression of angiogenesis or cell proliferation in tumors may cause the observed therapeutic effect. In conclusion, the results of the studies presented here support the hypothesis that the treatment of solid tumors with a combination of oncolytic virotherapy and immunotherapy has an additive effect over each treatment alone. Moreover, expression of the immunotherapeutic antibody by the oncolytic VACV locally in the tumor enhances the antitumor effect over systemic treatment with the same antibody. Combined, these results indicate that therapy with oncolytic VACVs expressing-therapeutic antibodies may be a promising approach for the treatment of cancer.},
  subject      = {Vaccinia-Virus},
  language  = {en}
}
@phdthesis{Banaszek2013,
  author    = {Banaszek, Agnes},
  title     = {Dual Antigen-Restricted Complementation of a Two-Part Trispecific Antibody for Targeted Immunotherapy of Blood Cancer},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-90174},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Cancer cells frequently escape from immune surveillance by down-regulating two important components of the immune defence: antigen-presenting MHC and costimulatory molecules. Therefore several novel anti-tumour compounds that aim to assist the immune system in recognising and fighting cancer are currently under development. Recombinant bispecific antibodies represent one group of such novel therapeutics. They target two different antigens and recruit cytotoxic effector cells to tumour cells. For cancer immunotherapy, bispecific T cell-engaging antibodies are already well characterised. These antibodies target a tumour-associated antigen and CD3ε, the constant molecule of the T cell receptor complex. On the one hand, this study presents the development of a bispecific antibody targeting CD3ε and the rhabdomyosarcoma-associated fetal acetylcholine receptor. On the other hand, it describes a novel two-part trispecific antibody format for the treatment of leukaemia and other haematological malignancies in the context of haematopoietic stem cell transplantation (HSCT). For HSCT, an HLA-identical donor is preferred, but very rarely available. In an HLA-mismatched setting, the HLA disparity could be exploited for targeted cancer treatment. In the present study, a two-part trispecific HLA-A2 × CD45 × CD3 antibody was developed for potential cases in which the patient is HLA-A2-positive, but the donor is not. This holds true for about half the cases in Germany, since HLA-A2 is the most common HLA molecule found here. Combinatorial targeting of HLA-A2 and the leucocyte-common antigen CD45 allows for highly specific dual-antigen restricted tumour targeting. More precisely, two single-chain antibody constructs were developed: i) a single-chain variable fragment (scFv) specific for HLA-A2, and ii) a scFv against CD45, both linked to the VL and the VH domain of a CD3ε-specific antibody, respectively. It turned out that, after the concomitant binding of these constructs to the same HLA-A2- and CD45-expressing cell, the unpaired variable domains of a CD3ε-specific antibody assembled to a functional scFv. In a therapeutic situation, this assembly should exclusively occur on the recipient's blood cancer cells, leading to T cell-mediated cancer cell destruction. In this way, a relapse of disease might be prevented, and standard therapy (radiation and chemotherapy) might be omitted. For both approaches, the antibody constructs were periplasmically expressed in E. coli, purified via His tag, and biochemically characterised. Their binding to the respective targets was proven by flow cytometry. The stimulatory properties of the antibodies were assayed by measuring IL-2 release after incubation with T cells and antigen-expressing target cells. Both the bispecific antibody against rhabdomyosarcoma and the assembled trispecific antibody against blood cancer mediated T-cell activation in a concentration-dependent manner at nanomolar concentrations. For the trispecific antibody, this effect indeed proved to be dual antigen-restricted, as it could be blocked by prior incubation of either HLA-A2- or CD45-specific scFv and did not occur on single-positive (CD45+) or double-negative (HLA-A2- CD45-) target cells. Furthermore, antibodies from both approaches recruited T cells for tumour cell destruction in vitro.},
  subject      = {Immuntherapie},
  language  = {en}
}