title: A new anti-mesothelin antibody targets selectively the membrane-associated form.
abstract: Mesothelin is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that shows promise as a target for antibody-directed cancer therapy. High levels of soluble forms of the antigen represent a barrier to directing therapy to cellular targets. The ability to develop antibodies that can selectively discriminate between membrane-bound and soluble conformations of a specific protein, and thus target only the membrane-associated antigen, is a substantive issue. We show that use of a tolerance protocol provides a route to such discrimination. Mice were tolerized with soluble mesothelin and a second round of immunizations was performed using mesothelin transfected P815 cells. RNA extracted from splenocytes was used in phage display to obtain mesothelin-specific antigen-binding fragments (Fabs) that were subsequently screened by flow cytometry and ELISA. This approach generated 147 different Fabs in 34 VH-CDR3 families. Utilizing competition assays with soluble protein and mesothelin-containing serum obtained from metastatic cancer patients, 10 of these 34 VH-CDR3 families were found to bind exclusively to the membrane-associated form of mesothelin. Epitope mapping performed for the 1H7 clone showed that it does not recognize GPI anchor. VH-CDR3 sequence analysis of all Fabs showed significant differences between Fabs selective for the membrane-associated form of the antigen and those that recognize both membrane bound and soluble forms. This work demonstrates the potential to generate an antibody specific to the membrane-bound form of mesothelin. 1H7 offers potential for therapeutic application against mesothelin-bearing tumors, which would be largely unaffected by the presence of the soluble antigen.
authors.affiliation: a University of Bourgogne-Franche-Comte , Besançon Cedex , France.|b ITAC Platform of Clinical Investigation Center-Biotherapy , Besançon Cedex , France.
authors.forename: Kamal
authors.initials: K
authors.lastname: Asgarov
authors.affiliation: a University of Bourgogne-Franche-Comte , Besançon Cedex , France.|b ITAC Platform of Clinical Investigation Center-Biotherapy , Besançon Cedex , France.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Balland
authors.affiliation: a University of Bourgogne-Franche-Comte , Besançon Cedex , France.|b ITAC Platform of Clinical Investigation Center-Biotherapy , Besançon Cedex , France.
authors.forename: Charline
authors.initials: C
authors.lastname: Tirole
authors.affiliation: a University of Bourgogne-Franche-Comte , Besançon Cedex , France.|b ITAC Platform of Clinical Investigation Center-Biotherapy , Besançon Cedex , France.
authors.forename: Adeline
authors.initials: A
authors.lastname: Bouard
authors.affiliation: a University of Bourgogne-Franche-Comte , Besançon Cedex , France.|c Blood Bank Bourgogne-Franche-comté , Porto , Portugal.
authors.forename: Virginie
authors.initials: V
authors.lastname: Mougey
authors.affiliation: d Fairjourney Biologics , Porto , Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Ramos
authors.affiliation: d Fairjourney Biologics , Porto , Portugal.
authors.forename: António
authors.initials: A
authors.lastname: Barroso
authors.affiliation: b ITAC Platform of Clinical Investigation Center-Biotherapy , Besançon Cedex , France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Zangiacomi
authors.affiliation: e J.Minjoz University Hospital , Besançon Cedex , France.
authors.forename: Marine
authors.initials: M
authors.lastname: Jary
authors.affiliation: e J.Minjoz University Hospital , Besançon Cedex , France.
authors.forename: Stefano
authors.initials: S
authors.lastname: Kim
authors.affiliation: d Fairjourney Biologics , Porto , Portugal.
authors.forename: Maria
authors.initials: M
authors.lastname: Gonzalez-Pajuelo
authors.affiliation: a University of Bourgogne-Franche-Comte , Besançon Cedex , France.|b ITAC Platform of Clinical Investigation Center-Biotherapy , Besançon Cedex , France.|e J.Minjoz University Hospital , Besançon Cedex , France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Royer
authors.affiliation: f arGEN-X BVBA , Zwijnaarde , Belgium.
authors.forename: Hans
authors.initials: H
authors.lastname: de Haard
authors.affiliation: g Mabdesigns , London , UK.
authors.forename: Andy
authors.initials: A
authors.lastname: Clark
authors.affiliation: g Mabdesigns , London , UK.
authors.forename: John
authors.initials: J
authors.lastname: Wijdenes
authors.affiliation: a University of Bourgogne-Franche-Comte , Besançon Cedex , France.|b ITAC Platform of Clinical Investigation Center-Biotherapy , Besançon Cedex , France.|c Blood Bank Bourgogne-Franche-comté , Porto , Portugal.|e J.Minjoz University Hospital , Besançon Cedex , France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Borg
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology; D058851:GPI-Linked Proteins / Q000276:immunology*; D006801:Humans; D007108:Immune Tolerance; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D000090204:Mesothelin; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D058851:GPI-Linked Proteins; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins; C000715528:Msln protein, mouse; D000090204:Mesothelin
keywords: Competition assay; membrane-specific antibody; mesothelin; phage display; serum mesothelin; soluble mesothelin; therapeutic antibody; tolerance immunization