title: Process development of a human recombinant diabody expressed in E. coli: engagement of CD99-induced apoptosis for target therapy in Ewing's sarcoma.
abstract: Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60 % soluble fraction, we obtained a yield of 25 mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75 % soluble fraction 35 mg C7 dAbd per L of cell culture grown in a synthetic media containing 3 g/L of vegetable peptone and 1 g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells.
authors.affiliation: Diatheva S.R.L, via T.Campanella 1, 61032, Fano, Italy. d.moricoli@diatheva.com.
authors.forename: Diego
authors.initials: D
authors.lastname: Moricoli
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: Sabrina
authors.initials: S
authors.lastname: Dominici
authors.affiliation: Diatheva S.R.L, via T.Campanella 1, 61032, Fano, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Fiori
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: 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: Clara
authors.initials: C
authors.lastname: Guerzoni
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: Maria Cristina
authors.initials: MC
authors.lastname: Manara
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: Michela
authors.initials: M
authors.lastname: Pasello
authors.affiliation: Diatheva S.R.L, via T.Campanella 1, 61032, Fano, Italy.
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Laguardia
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. katia.scotlandi@ior.it.|PROMETEO Laboratory, STB, RIT Department, Istituto Ortopedico Rizzoli, Bologna, 40136, Italy. katia.scotlandi@ior.it.
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 / Q000037:antagonists & inhibitors*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001859:Bone Neoplasms / Q000188:drug therapy; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology*; D012512:Sarcoma, Ewing / Q000188:drug therapy; D057127:Single-Chain Antibodies / Q000235:genetics / Q000494:pharmacology*
chemical_list: D000071443:12E7 Antigen; D000970:Antineoplastic Agents; C061828:CD99 protein, human; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: CD99; E. coli; Ewing’s sarcoma; Human bivalent diabody; Large-scale manufacturing; Process development