title: Generation of monoclonal antibody MS17-57 targeting secreted alkaline phosphatase ectopically expressed on the surface of gastrointestinal cancer cells.
abstract: BACKGROUND
  Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is crucial for successful drug development. However, due to immune tolerance, making it difficult to generate antibodies using conventional approaches.
  
  
  RESULTS
  Mixed four human gastric cancer (GC) cell lines were used as the immunogen in A/J mice; sixteen highly positive hybridoma colonies were selected via fluorescence-activated cell sorting-high throughput screening (FACS-HTS) using a total of 20,000 colonies in sixty-seven 96-well plates against live cells (mixed human GC cells versus human PBMC controls). MS17-57 and control commercial Alkaline Phosphatase (ALP) mAbs were used to confirm the target antigens (Ags), which were identified as ALPs expressed on the GC cell surface through a combination of western blot, immunoprecipitation and mass spectrometry (MS). MS identified the Ags recognized by MS17-57 to be two variants of a secreted ALP, PALP and IALP (Placental and intestinal ALP). These proteins belong to a hydrolase enzyme family responsible for removing phosphate groups from many types of molecules. Immunofluorescence staining using MS17-57 demonstrated higher staining of gastrointestinal (GI) cancer tissues compared to normal GI tissues (P<0.03), and confirmed binding of MS17-57 to be restricted to a functional epitope expressed on the cancer cell surface. Proliferation assays using the PALP/IALP-expressing GC cell lines demonstrated that MS17-57 inhibited cell growth by 32 ± 8%. Transwell cell migration assays documented that MS17-57 can inhibit PALP/IALP-expressing GI cancer cell migration by 25 ± 5%. MS17-57 mAb inhibited tumor growth in nude mice.
  
  
  CONCLUSIONS
  Our findings indicate that PALP and IALP can be ectopically expressed on extracellular matrix of GI cancers, and that MS17-57 directed against PALP/IALP can inhibit GI cancer cells growth and migration in vitro and in vivo. This investigation provides an example of identification of cancer biomarkers representing promising therapeutic targets using mAb generated through a novel HTS technology.
authors.affiliation: Department of Pathology, Nanjing Medical University, Nanjing, Jiangsu Province, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.forename: Jianpeng
authors.initials: J
authors.lastname: Gao
authors.forename: Runhua
authors.initials: R
authors.lastname: Feng
authors.forename: Yuling
authors.initials: Y
authors.lastname: Wang
authors.forename: Xuehua
authors.initials: X
authors.lastname: Chen
authors.forename: Jianyu
authors.initials: J
authors.lastname: Sun
authors.forename: Dongqing
authors.initials: D
authors.lastname: Zhang
authors.forename: Zhenggang
authors.initials: Z
authors.lastname: Zhu
authors.forename: Lee M
authors.initials: LM
authors.lastname: Ellis
authors.forename: Mason
authors.initials: M
authors.lastname: Lu
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Lee
authors.forename: Zhenqing
authors.initials: Z
authors.lastname: Feng
authors.forename: Bingya
authors.initials: B
authors.lastname: Liu
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D001483:Base Sequence; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D005770:Gastrointestinal Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D058990:Molecular Targeted Therapy / Q000379:methods*; D015398:Signal Transduction
chemical_list: D000911:Antibodies, Monoclonal; D000469:Alkaline Phosphatase