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Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type

Steven Rust1*, Sandrine Guillard1, Kris Sachsenmeier2, Carl Hay2, Max Davidson3, Anders Karlsson3, Roger Karlsson3, Erin Brand2, David Lowne1, John Elvin1, Matt Flynn2, Gene Kurosawa4, Robert Hollingsworth2, Lutz Jermutus1 and Ralph Minter1

Author Affiliations

1 MedImmune, Granta Park, Cambridge, CB21 6GH, UK

2 MedImmune, One MedImmune Way, Gaithersburg, MD, 20287, US

3 Nanoxis AB, Lennart Torstenssonsgatan 5, SE-40016, Gothenburg, Sweden

4 Division of Antibody Project, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan

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Molecular Cancer 2013, 12:11  doi:10.1186/1476-4598-12-11

Published: 13 February 2013



The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options.


The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model.


All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated using the anti-CD73 antibody, which bound other ‘triple negative’ breast cancer cell lines and produced significant tumour growth inhibitory activity in a MDA-MB-231 xenograft model.


This study has demonstrated that multiple methods are required to successfully analyse the membranome of a desired cell type. It has also successfully demonstrated that phenotypic antibody screening provides a mechanism for rapidly discovering and evaluating antibody tractable targets, which can significantly accelerate the therapeutic discovery process.

Phage display; Hybridoma; Antibody; Phenotypic screening; Membranome; CD73