title: Combining phenotypic and proteomic approaches to identify membrane targets in a 'triple negative' breast cancer cell type.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: MedImmune, Granta Park, Cambridge, CB21 6GH, UK. rusts@medimmune.com
authors: Steven
authors: S
authors: Rust
authors: Sandrine
authors: S
authors: Guillard
authors: Kris
authors: K
authors: Sachsenmeier
authors: Carl
authors: C
authors: Hay
authors: Max
authors: M
authors: Davidson
authors: Anders
authors: A
authors: Karlsson
authors: Roger
authors: R
authors: Karlsson
authors: Erin
authors: E
authors: Brand
authors: David
authors: D
authors: Lowne
authors: John
authors: J
authors: Elvin
authors: Matt
authors: M
authors: Flynn
authors: Gene
authors: G
authors: Kurosawa
authors: Robert
authors: R
authors: Hollingsworth
authors: Lutz
authors: L
authors: Jermutus
authors: Ralph
authors: R
authors: Minter
mesh_terms: D015720:5'-Nucleotidase / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D058851:GPI-Linked Proteins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D006825:Hybridomas; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D019151:Peptide Library; D010641:Phenotype; D011485:Protein Binding; D020543:Proteome / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D054789:Ribosome Inactivating Proteins, Type 1 / Q000378:metabolism / Q000494:pharmacology; D000076985:Saporins; D057127:Single-Chain Antibodies / Q000378:metabolism* / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
chemical_list: D015720:5'-Nucleotidase; D000972:Antineoplastic Agents, Phytogenic; D014408:Biomarkers, Tumor; D004337:Drug Carriers; D058851:GPI-Linked Proteins; D008565:Membrane Proteins; D019151:Peptide Library; D020543:Proteome; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D057127:Single-Chain Antibodies; C508053:ERBB2 protein, human; C548241:NT5E protein, human