title: Monoclonal antibody MX35 detects the membrane transporter NaPi2b (SLC34A2) in human carcinomas.
abstract: Mouse monoclonal antibody MX35 was developed against ovarian cancer. The antibody showed homogeneous reactivity with approximately 90% of human ovarian epithelial cancers and with a limited number of normal tissues by immunohistochemistry. Although mAb MX35 has been used in a number of clinical trials in ovarian cancer, it has been difficult to define the molecular identity of MX35. We report here that mAb MX35 recognizes the sodium-dependent phosphate transport protein 2b (NaPi2b) in human cancer cells. This conclusion is based on several lines of experimental evidence, including 1) the identification of SLC34A2, the gene coding for NaPi2b, by immunoscreening an ovarian cancer cell line cDNA expression library with mAb MX35; 2) mass spectrometry sequencing of peptides obtained by fragmentation from mAb MX35 affinity-purified antigen, which show complete sequence homology to amino acid sequences in NaPi2b; 3) selective down-regulation of SLC34A2 gene expression by RNA interference and the resulting loss of mAb MX35 binding to MX35-expressing human cancer cells; and 4) the demonstration of specific mAb MX35 reactivity with recombinant fusion proteins and with synthetic peptides of the putative largest extracellular loop of NaPi2b. We further show that NaPi2b in cancer cells is expressed on the cell surface as a heavily N-glycosylated protein, with evidence of additional post-translational modifications such as palmitoylation and the formation of disulfide bridges in the major extracellular loop. Membrane transporter molecules, such as NaPi2b, represent a new family of potential cell surface targets for the immunotherapy of cancer with monoclonal antibodies.
authors.affiliation: Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Beatrice W T
authors.initials: BW
authors.lastname: Yin
authors.forename: Ramziya
authors.initials: R
authors.lastname: Kiyamova
authors.forename: Ramon
authors.initials: R
authors.lastname: Chua
authors.forename: Otavia L
authors.initials: OL
authors.lastname: Caballero
authors.forename: Ivan
authors.initials: I
authors.lastname: Gout
authors.forename: Vitalina
authors.initials: V
authors.lastname: Gryshkova
authors.forename: Nimesh
authors.initials: N
authors.lastname: Bhaskaran
authors.forename: Serhiy
authors.initials: S
authors.lastname: Souchelnytskyi
authors.forename: Ulf
authors.initials: U
authors.lastname: Hellman
authors.forename: Valeriy
authors.initials: V
authors.lastname: Filonenko
authors.forename: Achim A
authors.initials: AA
authors.lastname: Jungbluth
authors.forename: Kunle
authors.initials: K
authors.lastname: Odunsi
authors.forename: Kenneth O
authors.initials: KO
authors.lastname: Lloyd
authors.forename: Lloyd J
authors.initials: LJ
authors.lastname: Old
authors.forename: Gerd
authors.initials: G
authors.lastname: Ritter
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007167:Immunotherapy / Q000639:trends; D013058:Mass Spectrometry; D051379:Mice; D010051:Ovarian Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D034741:RNA, Small Interfering; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb