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

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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.

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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.

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Authors and Affiliations

  1. Diatheva S.R.L, via T.Campanella 1, 61032, Fano, Italy

    Diego Moricoli, Damiano Cosimo Carbonella, Sabrina Dominici, Valentina Fiori, Maria Cristina Balducci & Maria Elena Laguardia

  2. Department of Biomolecular Science, Section of Biochemistry and Molecular Biology University of Urbino “Carlo Bo”, Via Saffi 2, 61029, Urbino, Italy

    Mauro Magnani

  3. Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy

    Maurizio Cianfriglia

  4. CRS Development of Biomolecular Therapies, Experimental Oncology Laboratory, Istituto Ortopedico Rizzoli, Bologna, 40136, Italy

    Clara Guerzoni, Maria Cristina Manara, Michela Pasello & Katia Scotlandi

  5. PROMETEO Laboratory, STB, RIT Department, Istituto Ortopedico Rizzoli, Bologna, 40136, Italy

    Clara Guerzoni, Maria Cristina Manara, Michela Pasello & Katia Scotlandi

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  1. Diego Moricoli
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  2. Damiano Cosimo Carbonella
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  9. Maria Elena Laguardia
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  11. Katia Scotlandi
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Correspondence to Diego Moricoli or Katia Scotlandi.

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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.

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Moricoli, D., Carbonella, D.C., Dominici, S. et al. Process development of a human recombinant diabody expressed in E. coli: engagement of CD99-induced apoptosis for target therapy in Ewing’s sarcoma. Appl Microbiol Biotechnol 100, 3949–3963 (2016). https://doi.org/10.1007/s00253-015-7226-5

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