title: Editor's Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response.
abstract: Protein aggregation is associated with enhanced immunogenicity of biotherapeutics. As a result, regulatory guidelines recommend screening for aggregation during bioprocessing. However, the mechanisms underlying the enhanced immunogenicity of aggregates are poorly understood. In the investigations described herein, the immunogenicity in mice of a humanized single chain variable antibody fragment (scFv) purified after expression in Escherichia coli has been examined. Reproducible scFv aggregates were obtained within the subvisible particle size range (mean diameter 2 µm) using thermal and mechanical stresses. Intraperitoneal immunization of BALB/c strain mice with 1 mg/ml of aggregated or monomeric scFv induced similar IgG and IgG1 antibody responses. In contrast, aggregate preparations stimulated significantly higher levels of anti-scFv IgG2a antibody than did the monomer. In comparative studies, aggregates of ovalbumin (OVA) within the subvisible particle size range were prepared by stir stress, and their immunogenicity compared with that of monomeric OVA in mice. Aggregated and monomeric OVA induced similar anti-OVA IgG and IgG1 antibody responses, whereas IgG2a antibody levels were significantly higher in aggregate-immunized mice. Furthermore, cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were consistent with aggregated preparations inducing a T helper (Th) 1-type response. Aggregated proteins within the subvisible range were therefore shown to induce a preferential Th1 type response, whereas monomeric proteins elicited a selective Th2 response. These data indicate that protein aggregation can impact on both the vigor and quality of immune responses.
authors.affiliation: *Faculty of Life Sciences, The University of Manchester, C1256, Michael Smith Building, Dover St, Manchester M13 9PT, UK;
authors.forename: Kirsty D
authors.initials: KD
authors.lastname: Ratanji
authors.affiliation: *Faculty of Life Sciences, The University of Manchester, C1256, Michael Smith Building, Dover St, Manchester M13 9PT, UK;
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Dearman
authors.affiliation: *Faculty of Life Sciences, The University of Manchester, C1256, Michael Smith Building, Dover St, Manchester M13 9PT, UK;
authors.forename: Ian
authors.initials: I
authors.lastname: Kimber
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG.
authors.forename: Robin
authors.initials: R
authors.lastname: Thorpe
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG.
authors.forename: Meenu
authors.initials: M
authors.lastname: Wadhwa
authors.affiliation: *Faculty of Life Sciences, The University of Manchester, C1256, Michael Smith Building, Dover St, Manchester M13 9PT, UK; jeremy.derrick@manchester.ac.uk.
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Derrick
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000096:biosynthesis / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008807:Mice, Inbred BALB C; D010047:Ovalbumin / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D013154:Spleen / Q000166:cytology; D018417:Th1 Cells / Q000276:immunology*
chemical_list: D016207:Cytokines; D011506:Proteins; D010047:Ovalbumin
keywords: OVA.; biotherapeutic; immunogenicity; protein aggregation; scFv