title: Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.
abstract: BACKGROUND
  30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).
  
  
  RESULTS
  With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.
  
  
  CONCLUSIONS
  Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.
authors.affiliation: Department of Biomedical Sciences, Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria; Christian Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Katrin
authors.initials: K
authors.lastname: Spiesberger
authors.affiliation: Christian Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Florian
authors.initials: F
authors.lastname: Paulfranz
authors.affiliation: Christian Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Anton
authors.initials: A
authors.lastname: Egger
authors.affiliation: Christian Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Judith
authors.initials: J
authors.lastname: Reiser
authors.affiliation: Department of Biomedical Sciences, Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Claus
authors.initials: C
authors.lastname: Vogl
authors.affiliation: Christian Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Judith
authors.initials: J
authors.lastname: Rudolf-Scholik
authors.affiliation: Department of Biomedical Sciences, Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria; Department of Agrobiotechnology (IFA Tulln), Institute of Biotechnology in Animal Production, University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria.
authors.forename: Corina
authors.initials: C
authors.lastname: Mayrhofer
authors.affiliation: Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, Germany.
authors.forename: Ludger
authors.initials: L
authors.lastname: Grosse-Hovest
authors.affiliation: Department of Biomedical Sciences, Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria; Christian Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine Vienna, Vienna, Austria; Department of Agrobiotechnology (IFA Tulln), Institute of Biotechnology in Animal Production, University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria.
authors.forename: Gottfried
authors.initials: G
authors.lastname: Brem
mesh_terms: D000328:Adult; D000818:Animals; D030801:Animals, Genetically Modified; D018033:Antibodies, Bispecific / Q000276:immunology*; D018106:CD28 Antigens / Q000276:immunology; D002417:Cattle / Q000235:genetics / Q000276:immunology*; D045744:Cell Line, Tumor; D011508:Chondroitin Sulfate Proteoglycans / Q000276:immunology*; D006801:Humans; D008297:Male; D008545:Melanoma / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000276:immunology*; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D018033:Antibodies, Bispecific; D018106:CD28 Antigens; D011508:Chondroitin Sulfate Proteoglycans; D008565:Membrane Proteins; D057127:Single-Chain Antibodies; C101390:CSPG4 protein, human; D000099216:Chondroitin Sulfate Proteoglycan 4