title: Development of a Trispecific Antibody Designed to Simultaneously and Efficiently Target Three Different Antigens on Tumor Cells.
abstract: Targeting Eph (erythropoietin producing hepatoma) receptors with monoclonal antibodies is being explored as therapy for several types of cancer. To test whether simultaneous targeting of EphA2, EphA4, and EphB4 would be an effective approach to cancer therapy, we generated a recombinant trispecific antibody using the variable domain genes of anti-EphA2, anti-EphA4, and anti-EphB4 monoclonal antibodies. A multidisciplinary approach combining biochemical, biophysical, and cellular-based assays was used to characterize the trispecific antibody in vitro and in vivo. Here we demonstrate that the trispecific antibody is expressed at high levels by mammalian cells, monodispersed in solution, thermostable, capable of simultaneously binding the three receptors, and able to activate the three targets effectively as evidenced by receptor internalization and degradation both in vitro and in vivo. Furthermore, pharmacokinetic analysis using tumor-bearing nude mice showed that the trispecific antibody remains in the circulation similarly to its respective parental antibodies. These results indicate that simultaneous blockade of EphA2, EphA4, and EphB4 could be an attractive approach to cancer therapy.
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Nazzareno
authors.initials: N
authors.lastname: Dimasi
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Ryan
authors.initials: R
authors.lastname: Fleming
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Carl
authors.initials: C
authors.lastname: Hay
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Rob
authors.initials: R
authors.lastname: Woods
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Linda
authors.initials: L
authors.lastname: Xu
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Herren
authors.initials: H
authors.lastname: Wu
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Changshou
authors.initials: C
authors.lastname: Gao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000941:Antigens / Q000276:immunology*; D002152:Calorimetry, Differential Scanning; D015195:Drug Design; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D036104:Receptor, EphA2 / Q000037:antagonists & inhibitors* / Q000276:immunology; D036122:Receptor, EphA4 / Q000037:antagonists & inhibitors* / Q000276:immunology; D036224:Receptor, EphB4 / Q000037:antagonists & inhibitors* / Q000276:immunology; D020349:Surface Plasmon Resonance; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D036104:Receptor, EphA2; D036122:Receptor, EphA4; D036224:Receptor, EphB4
keywords: EphA2; EphA4; EphB4; analytical ultracentrifugation; differential scanning calorimetry; trispecific antibody; tumor targeting