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Development of Oligoclonal Nanobodies for Targeting the Tumor-Associated Glycoprotein 72 Antigen

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Abstract

The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies that specifically recognize the TAG-72 antigen. All selected nanobodies were shown to selectively bind to this cancer-related molecule with low-nanomolar affinities and do not cross-react with other antigens, such as MUC1 or HER2. Furthermore, they can detect TAG-72 in concentrations as low as 5 U/ml which is valuable in sensitive detection of this molecule in cancerous patients. Cell ELISA experiments proved their ability for binding to the native target antigen on TAG-72 expressing cells while not showing any reactivity to HT-29 cells, a TAG-72-negative cell line. Using competition studies, we found that each nanobody recognizes a distinct epitope on the TAG-72 antigen that is different from the one recognized by the mouse anti-TAG-72 antibody, CC49. Considering their high specificity, reduced immunogenicity and multi-targeting behavior, these oligoclonal nanobodies represent a promising tool to target TAG-72 over-expressing tumor cells.

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Abbreviations

CDR:

Complementari determining region

Fab:

Fragment antigen binding

FR:

Framework region

HAMA:

Human anti-mouse antibody

IPTG:

Isopropyl-β-d-thiogalactopyranoside

scFv:

Single chain variable fragment

mAb:

Monoclonal antibody

TAG-72:

Tumor-associated glycoprotein 72

TMB:

Tetramethylbenzidine

VHH:

Variable heavy-chain domain of heavy-chain antibody

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Acknowledgments

We are grateful to Dr. Oliver Jay Broom (KIPA—Krahbichler Intellectual Property Advisors AB, 251 10 Helsingborg, Sweden) and Dr. Ladan Parhamifar (Center of Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark) for their critical language revision of the manuscript. This study was supported by Pasteur Institute of Iran, Tehran, Iran and the Biotechnology committee of Tarbiat Modares University (TMU-88-8-67), Tehran, Iran.

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Authors and Affiliations

  1. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

    Zahra Sharifzadeh, Mohammad Ali Shokrgozar & Fatemeh Rahimi Jamnani

  2. Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

    Fatemeh Rahbarizadeh & Seyed Hamid Aghaee Bakhtiari

  3. School of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran

    Davoud Ahmadvand

  4. Department of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran

    Fereidoun Mahboudi

Authors
  1. Zahra Sharifzadeh
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  2. Fatemeh Rahbarizadeh
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  3. Mohammad Ali Shokrgozar
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  6. Fatemeh Rahimi Jamnani
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Correspondence to Fatemeh Rahbarizadeh.

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Sharifzadeh, Z., Rahbarizadeh, F., Shokrgozar, M.A. et al. Development of Oligoclonal Nanobodies for Targeting the Tumor-Associated Glycoprotein 72 Antigen. Mol Biotechnol 54, 590–601 (2013). https://doi.org/10.1007/s12033-012-9601-0

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  • DOI: https://doi.org/10.1007/s12033-012-9601-0

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