title: Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling.
abstract: BACKGROUND
  Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets.
  
  
  METHODS
  ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin.
  
  
  RESULTS
  Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model.
  
  
  CONCLUSIONS
  Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.
authors.affiliation: MedImmune, Granta Park, Cambridge, CB21 6GH, UK. sandercocka@medimmune.com.
authors.forename: Alan M
authors.initials: AM
authors.lastname: Sandercock
authors.affiliation: MedImmune, Granta Park, Cambridge, CB21 6GH, UK. rusts@medimmune.com.
authors.forename: Steven
authors.initials: S
authors.lastname: Rust
authors.affiliation: MedImmune, Granta Park, Cambridge, CB21 6GH, UK. guillards@medimmune.com.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Guillard
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20287, USA. sachsenmeierk@medimmune.com.
authors.forename: Kris F
authors.initials: KF
authors.lastname: Sachsenmeier
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20287, USA. holoweckyjn@medimmune.com.
authors.forename: Nick
authors.initials: N
authors.lastname: Holoweckyj
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20287, USA. hayc@medimmune.com.
authors.forename: Carl
authors.initials: C
authors.lastname: Hay
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20287, USA. flynnm@medimmune.com.
authors.forename: Matt
authors.initials: M
authors.lastname: Flynn
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20287, USA. huangq@MedImmune.com.
authors.forename: Qihui
authors.initials: Q
authors.lastname: Huang
authors.affiliation: OcellO, Leiden BioPartner Center, J. H Oortweg 21, 2333 CH, Leiden, The Netherlands. Kuan.yan@OcellO.nl.
authors.forename: Kuan
authors.initials: K
authors.lastname: Yan
authors.affiliation: OcellO, Leiden BioPartner Center, J. H Oortweg 21, 2333 CH, Leiden, The Netherlands. Bram.herpers@OcellO.nl.
authors.forename: Bram
authors.initials: B
authors.lastname: Herpers
authors.affiliation: OcellO, Leiden BioPartner Center, J. H Oortweg 21, 2333 CH, Leiden, The Netherlands. Leo.price@OcellO.nl.
authors.forename: Leo S
authors.initials: LS
authors.lastname: Price
authors.affiliation: Retrogenix, Crown House, Bingswood Estate, Whaley Bridge, High Peak, SK23 7LY, UK. Jo.Soden@retrogenix.com.
authors.forename: Jo
authors.initials: J
authors.lastname: Soden
authors.affiliation: Retrogenix, Crown House, Bingswood Estate, Whaley Bridge, High Peak, SK23 7LY, UK. Jim.freeth@retrogenix.com.
authors.forename: Jim
authors.initials: J
authors.lastname: Freeth
authors.affiliation: MedImmune, Granta Park, Cambridge, CB21 6GH, UK. jermutusl@medimmune.com.
authors.forename: Lutz
authors.initials: L
authors.lastname: Jermutus
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20287, USA. hollingsworthr@medimmune.com.
authors.forename: Robert
authors.initials: R
authors.lastname: Hollingsworth
authors.affiliation: MedImmune, Granta Park, Cambridge, CB21 6GH, UK. minterr@medimmune.com.
authors.forename: Ralph
authors.initials: R
authors.lastname: Minter
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001688:Biological Products / Q000494:pharmacology*; D014408:Biomarkers, Tumor; D002289:Carcinoma, Non-Small-Cell Lung; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D061505:Cell Surface Display Techniques; D002945:Cisplatin / Q000494:pharmacology; D004195:Disease Models, Animal; D055808:Drug Discovery / Q000379:methods*; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms; D051379:Mice; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D010641:Phenotype; D057127:Single-Chain Antibodies / Q000494:pharmacology; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D001688:Biological Products; D014408:Biomarkers, Tumor; C432044:CDCP1 protein, human; D015815:Cell Adhesion Molecules; D009363:Neoplasm Proteins; D019151:Peptide Library; D057127:Single-Chain Antibodies; D002945:Cisplatin