title: The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine.
abstract: Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with its neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. In addition, we reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8(+) T cell responses in two murine cancer models.
authors.affiliation: Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland.
authors.forename: Ziad
authors.initials: Z
authors.lastname: Julier
authors.affiliation: 1] Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland [2] World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Mikaël M
authors.initials: MM
authors.lastname: Martino
authors.affiliation: Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland.
authors.forename: Alexandre
authors.initials: A
authors.lastname: de Titta
authors.affiliation: Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland.
authors.forename: Laura
authors.initials: L
authors.lastname: Jeanbart
authors.affiliation: 1] Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland [2] Institute for Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland [3] Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637, USA [4] Materials Science Division, Argonne National Laboratory, Argonne, IL 60439, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Hubbell
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D019496:Cancer Vaccines / Q000276:immunology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D005337:Fibrin / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D008546:Melanoma, Experimental / Q000276:immunology / Q000473:pathology / Q000517:prevention & control; D051379:Mice; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000276:immunology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D012697:Serine Endopeptidases / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000819:agonists* / Q000378:metabolism; D014184:Transplantation, Homologous
chemical_list: D019496:Cancer Vaccines; D005353:Fibronectins; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D051197:Toll-Like Receptor 4; D005337:Fibrin; D010047:Ovalbumin; D012697:Serine Endopeptidases; C117309:pancreatic elastase II