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Gellini M.,Section of Pharmacogenetics | Ascione A.,Section of Pharmacogenetics | Flego M.,Section of Pharmacogenetics | Mallano A.,Section of Pharmacogenetics | And 8 more authors.
Current Pharmaceutical Biotechnology

The survival of pediatric patients with cancer entities including osteosarcoma and Ewing's sarcoma (ES), Remains extremely low hence novel treatment approaches are urgently needed. Therefore, based on the concept of targeted therapy, numerous potential targets for the treatment of these cancers have been evaluated pre-clinically or in some cases even clinically during the last decade. In ES the CD99 protein is an attractive target antigen. In this respect, a new entry site for therapeutic intervention may derive from specific human antibodies against CD99. Human scFvC7 was isolated from a semi-synthetic ETH-2 antibody phage library panned on the extracellular portion of recombinant human CD99 protein. The scFvC7 was genetically sequenced, tested for CD99 recognition on an array of recombinant CD99 fragments and measured for binding affinity by ELISA. Finally, it was tested for staining CD99 antigen on a large panel of tumor and normal cells and tissues by cytofluorimetric and immunohystochemical assays. The new antibody scFvC7 recognizes the CD99 extracellular domain included between residues 50 and 74 with a binding affinity of 2.4 x 10-8 M. In contrast with all other antibodies to CD99 so far isolated, scFvC7 shows a unique specificity in cancer cell recognition: It stained prevalently ES cells while no or weak reactivity was observed on the majority of the other tumor and normal cells and tissues. Thanks to its properties the new anti-CD99 antibody here described represents the first step towards the construction of new selective ES therapeutics. © 2013 Bentham Science Publishers. Source

Pasello M.,Rizzoli Orthopaedic Institute | Zamboni S.,Rizzoli Orthopaedic Institute | Mallano A.,Section of Pharmacogenetics | Flego M.,Section of Pharmacogenetics | And 3 more authors.
Journal of Biotechnology

Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2 × 109 individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated. © 2016 Elsevier B.V.. Source

Cianfriglia M.,Section of Pharmacogenetics | Mallano A.,Section of Pharmacogenetics | Ascione A.,Section of Pharmacogenetics | Dupuis M.L.,Section of Pharmacogenetics
International Journal of Oncology

In this study we elucidated the role of ATPbinding cassette (ABC) multi-drug transporter proteins and cellular factors such as Bcl-2 expression and CD33 downmodulation contributing to free and hP67.6 mAb linked calicheamicin-γ1 (CalC-γ1) resistance. We analyzed in a well designed HL60 cell system the relationship between the expression of ABC proteins, Bcl-2 and CD33 modulation with the activity of free and mAb-linked CalC-γ1. The results herein reported and discussed, strongly suggest that both MDR1-Pgp and MRP1 efflux systems are engaged by CalC-γ1, but only MDR1-Pgp over-expression efficiently abrogates drug cytotoxicity in MDR cells. Paradoxically, Bcl-2 expression, as observed for other anticancer compounds belonging to the enediyne family of drugs, confers CalC-γ1 susceptibility rather than resistance in HL60 cells. Further, the isolation of a resistant HL60 subline (HL60AL) that was developed by exposing the parental sensitive cells to subeffective doses of gemtuzumab ozogamicin (GO) over an extended period of time shows a reduced level of CD33 expression that represents an important escape mechanism of HL60 MDR cells to the cytotoxic effect of GO. Source

Cianfriglia M.,Section of Pharmacogenetics | Fiori V.,Diatheva srl | Dominici S.,Urbino University | Zamboni S.,Section of Pharmacogenetics | And 6 more authors.
Open Pharmacology Journal

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell surface glycoprotein involved in intercellular binding, belonging to the immunoglobulin superfamily. It is involved in cell-cell recognition and modulates cellular processes that range from vascular angiogenesis to the regulation of insulin homeostasis and T-cell proliferation. Aberrant expression of CEACAM1 is often associated with progression and metastatic potential in melanoma, lung carcinoma and other types of tumor. Tumor-specific antigens such as CEACAM1 are ideal targets for cancer immunotherapy because they are over-expressed by the cancer cell and not on non-malignant tissues, minimizing the risk of autoimmune destruction. Many of the limitations of therapeutic use of rodent monoclonal antibodies (mAbs) can now be overcome by exploiting the use of recombinant antibody fragments and the advances in antibody engineering methods to improve tumor retention, reduce immunogenicity and modulate pharmacokinetics. In addition, a novel effective model of immunotherapeutic treatments of tumors includes antibody drug conjugates (ADCs) that combine specific mAbs and antibody fragments with cytotoxic drugs, proteins, enzymes, radionuclides and nanoparticles. This review aims to describe how these antibody engineering approaches can meet the challenges for generating new and effective antibody constructs for diagnosis and therapy of CEACAM1 expressing malignancies. © Cianfriglia et al. Source

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