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Applied Microbiology and Biotechnology

, Volume 100, Issue 9, pp 3949–3963 | Cite as

Process development of a human recombinant diabody expressed in E. coli: engagement of CD99-induced apoptosis for target therapy in Ewing’s sarcoma

  • Diego MoricoliEmail author
  • Damiano Cosimo Carbonella
  • Sabrina Dominici
  • Valentina Fiori
  • Maria Cristina Balducci
  • Clara Guerzoni
  • Maria Cristina Manara
  • Michela Pasello
  • Maria Elena Laguardia
  • Maurizio Cianfriglia
  • Katia ScotlandiEmail author
  • Mauro Magnani
Biotechnological products and process engineering

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.

Keywords

Human bivalent diabody E. coli Ewing’s sarcoma CD99 Process development Large-scale manufacturing 

Notes

Acknowledgments

We are thankful to Dr. Lisa Pucci (Diatheva) for residual DNA analysis and Dr. Claudia Gabucci (University of Urbino) and Dr. Luca Micci (Emory University) for critical reading of the manuscript.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

This study was supported by grant from the Italian Association for Cancer Research (Katia Scotlandi—AIRC Project N.14049).

Conflict of interest

Diego Moricoli, Damiano Cosimo Carbonella, Sabrina Dominici, Valentina Fiori, Maria Cristina Balducci and Maria Elena Laguardia are Diatheva s.r.l employees. Mauro Magnani has Diatheva stock and/or warrants. No potential conflicts of interest were disclosed by the other authors.

Supplementary material

253_2015_7226_MOESM1_ESM.pdf (389 kb)
ESM 1 (PDF 388 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Diego Moricoli
    • 1
    Email author
  • Damiano Cosimo Carbonella
    • 1
  • Sabrina Dominici
    • 1
  • Valentina Fiori
    • 1
  • Maria Cristina Balducci
    • 1
  • Clara Guerzoni
    • 4
    • 5
  • Maria Cristina Manara
    • 4
    • 5
  • Michela Pasello
    • 4
    • 5
  • Maria Elena Laguardia
    • 1
  • Maurizio Cianfriglia
    • 3
  • Katia Scotlandi
    • 4
    • 5
    Email author
  • Mauro Magnani
    • 2
  1. 1.Diatheva S.R.LFanoItaly
  2. 2.Department of Biomolecular ScienceSection of Biochemistry and Molecular Biology University of Urbino “Carlo Bo”UrbinoItaly
  3. 3.Department of Therapeutic Research and Medicines EvaluationIstituto Superiore di SanitàRomeItaly
  4. 4.CRS Development of Biomolecular Therapies, Experimental Oncology LaboratoryIstituto Ortopedico RizzoliBolognaItaly
  5. 5.PROMETEO Laboratory, STB, RIT DepartmentIstituto Ortopedico RizzoliBolognaItaly

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