Secreted alkaline phosphatase defined by ms17-57 mab and ectopically expressed on the surface of GI cancer cells as a potential therapeutic target.

Abstract

e22169 Background: There remains considerable interest in generating monoclonal antibody (mAb) directed against specific tumor targets for antigen (Ag) discovery, diagnosis and therapy. In this investigation, mAbs were generated against potentially novel Ags on the cancer cell surface using a flow cytometry-high throughput screening (FACS-HTS) strategy. Methods: Mixed live cells from four human gastric cancer (GC) cell lines were used as the immunogen in A/J mice; Sixteen highly positive hybridoma colonies were selected via FACS-HTS using a total of 20,000 colonies in sixty-seven 96-well plates against live cells (mixed human GC cells versus normal human PBMC). The purified ms17-57 and control commercial ALP mAbs were used to confirm the target Ags, which were identified as alkaline phosphatases (ALPs) expressed on the GC cell surface through a combination of western blot, immunoprecipitation and mass spectrometry (MS). Results: 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 (i.e. dephosphorylation). Immunohistochemical staining using ms17-57 demonstrated significantly higher staining of gastrointestinal (GI; gastric and colorectal) cancer tissues than normal (non-cancer) GI control tissues (P<0.01), and confirmed binding of ms17-57 to be restricted to a functional epitope expressed on the cancer cell surface. Proliferation assay using the PALP/IALP-expressing GC cell lines demonstrated that ms17-57 inhibited cell growth by 32±8%. Transwell cell migration assay documented that ms17-57 can inhibit PALP/IALP-expressing GI cancer cell migration. Conclusions: Our findings indicate that PALP and IALP can be ectopically expressed in the extracellular matrix of GI cancers, and that ms17-57 directed against PALP/IALP identified through HTS can inhibit GI cancer cell growth and migration. This investigation provides an example of identification of cancer biomarkers representing promising therapeutic targets using mAb generated through a novel HTS technology.