title: Conversion of murine antibodies to human antibodies and their optimization for ovarian cancer therapy targeted to the folate receptor.
abstract: We previously developed murine and chimeric antibodies against a specific epithelial ovarian carcinoma (EOC) marker, named folate receptor (FR), and promising results were obtained in phase II trials. More recently, we successfully generated a completely human Fab fragment, C4, by conversion of one of the murine anti-FR antibodies to human antibody using phage display and guided selection. However, subsequent efforts to obtain C4 in a dimer format, which seems especially desirable for EOC locoregional treatment, resulted in a highly heterogeneous product upon natural dimerization and in a very poor production yield upon chemical dimerization by a non-hydrolyzable linker to a di-Fab-maleimide (DFM). We therefore designed, constructed and characterized a large Fab dual combinatorial human antibody phage display library obtained from EOC patients and potentially biased toward an anti-tumor response in an effort to obtain new anti-FR human antibodies suitable for therapy. Using this library and guiding the selection on FR-expressing cells with murine/human antibody chains, we generated four new human anti-FR antibody (AFRA) Fab fragments, one of which was genetically and chemically manipulated to obtain a chemical dimer, designated AFRA-DFM5.3, with high yield production and the capability for purification scaled-up to clinical grade. Overall affinity of AFRA-DFM5.3 was in the 2-digit nanomolar range, and immunohistochemistry indicated that the reagent recognized the FR expressed on EOC samples. (131)I-AFRA-DFM5.3 showed high immunoreactivity, in vitro stability and integrity, and specifically accumulated only in FR-expressing tumors in subcutaneous preclinical in vivo models. Overall, our studies demonstrate the successful conversion of murine to completely human anti-FR antibodies through the combined use of antibody phage display libraries biased toward an anti-tumor response, guided selection and chain shuffling, and point to the suitability of AFRA5.3 for future clinical application in ovarian cancer.
authors.affiliation: Unit of Molecular Therapies, Department of Experimental Oncology and Laboratories, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133, Milan, Italy. mariangela.figini@istitutotumori.mi.it
authors.forename: Mariangela
authors.initials: M
authors.lastname: Figini
authors.forename: Franck
authors.initials: F
authors.lastname: Martin
authors.forename: Renata
authors.initials: R
authors.lastname: Ferri
authors.forename: Elena
authors.initials: E
authors.lastname: Luison
authors.forename: Elena
authors.initials: E
authors.lastname: Ripamonti
authors.forename: Alberto
authors.initials: A
authors.lastname: Zacchetti
authors.forename: Mimosa
authors.initials: M
authors.lastname: Mortarino
authors.forename: Vito
authors.initials: V
authors.lastname: Di Cioccio
authors.forename: Giovanni
authors.initials: G
authors.lastname: Maurizi
authors.forename: Marcello
authors.initials: M
authors.lastname: Allegretti
authors.forename: Silvana
authors.initials: S
authors.lastname: Canevari
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000276:immunology*; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007150:Immunohistochemistry; D007167:Immunotherapy / Q000379:methods*; D051379:Mice; D010051:Ovarian Neoplasms / Q000276:immunology*; D019151:Peptide Library; D011956:Receptors, Cell Surface / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000235:genetics / Q000276:immunology
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins