title: Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.
abstract: Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems.
authors.affiliation: Diatheva s.r.l. via T. Campanella 1, 61032 Fano, Italy. Electronic address: d.moricoli@diatheva.com.
authors.forename: Diego
authors.initials: D
authors.lastname: Moricoli
authors.affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: William Anthony
authors.initials: WA
authors.lastname: Muller
authors.affiliation: Diatheva s.r.l. via T. Campanella 1, 61032 Fano, Italy.
authors.forename: Damiano Cosimo
authors.initials: DC
authors.lastname: Carbonella
authors.affiliation: Diatheva s.r.l. via T. Campanella 1, 61032 Fano, Italy.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Balducci
authors.affiliation: Department of Biomolecular Science, Section of Biochemistry and Molecular Biology University of Urbino "Carlo Bo", Via Saffi 2, 61029 Urbino, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Dominici
authors.affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Watson
authors.affiliation: Diatheva s.r.l. via T. Campanella 1, 61032 Fano, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Fiori
authors.affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Evan
authors.initials: E
authors.lastname: Weber
authors.affiliation: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Cianfriglia
authors.affiliation: CRS Development of Biomolecular Therapies, Experimental Oncology Laboratory, Istituto Ortopedico Rizzoli, Bologna 40136, Italy; PROMETEO Laboratory, STB, RIT Department, Istituto Ortopedico Rizzoli, Bologna 40136, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Scotlandi
authors.affiliation: Department of Biomolecular Science, Section of Biochemistry and Molecular Biology University of Urbino "Carlo Bo", Via Saffi 2, 61029 Urbino, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
mesh_terms: D000071443:12E7 Antigen; D000918:Antibody Specificity; D015703:Antigens, CD / Q000276:immunology*; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors* / Q000276:immunology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000276:immunology; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology; D019897:Periplasm / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D057127:Single-Chain Antibodies / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D057705:Transendothelial and Transepithelial Migration / Q000187:drug effects*
chemical_list: D000071443:12E7 Antigen; D015703:Antigens, CD; C061828:CD99 protein, human; D015815:Cell Adhesion Molecules; D057127:Single-Chain Antibodies
keywords: Aggregates; Blocking monocyte transmigration; Periplasmic inclusion bodies; Purification; Single chain variable fragment (scFv)