title: A CD20 tandem-epitope immunogen elicits antibody in mice that binds murine cell surface CD20 and depletes splenic B cells in vivo.
abstract: CD20 is an important target for monoclonal antibody therapy of B-cell malignancies and for some autoimmune disorders. Though there is interest in evaluating the induction of active immunity to CD20 in the mouse model, the CD20 extracellular domain (ECD) has significant secondary and tertiary structure which make it difficult to target using peptide immunogens. We constructed, expressed, and purified a recombinant immunogen, CD20ECD-6, which displays six tandemly repeated copies of the C-terminus of the murine CD20 ECD covalently linked to maltose-binding protein. Analysis of the purified protein suggested a complex conformation as the protein migrated in significantly retarded fashion by SDS-PAGE analysis. Immunization of mice and rabbits with the CD20ECD-6 led to the induction of antibodies reactive with the C-terminal ECD peptide sequence by ELISA and more importantly, with native cell surface CD20 on the murine B-cell lymphomas, 38C13 and A20. Immunoprecipitation using the rabbit antisera and non-denaturing detergent confirmed the identity of the bound cell surface protein as murine CD20 and suggested that the cell-binding antibodies were specific for the native, folded conformation. Finally, immunization of mice with the CD20ECD-6 using Freund's or QS-21 adjuvants was shown to exert significant biological effects in vivo with the pronounced depletion of splenic B cells. The tandem-epitope immunogen represents a promising tool for eliciting and studying active autoimmunity to CD20, as a basis for potential development of new immunotherapeutics for cancer and autoimmune diseases.
authors.affiliation: Division of Hematology-Oncology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48105, USA.
authors.forename: Jon
authors.initials: J
authors.lastname: Oscherwitz
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Gribbin
authors.forename: Kemp B
authors.initials: KB
authors.lastname: Cease
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D018951:Antigens, CD20 / Q000737:chemistry / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D049109:Cell Proliferation; D000939:Epitopes / Q000276:immunology*; D005110:Extracellular Space / Q000276:immunology; D005260:Female; D015322:Gene Rearrangement, B-Lymphocyte; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013154:Spleen / Q000276:immunology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology
chemical_list: D000906:Antibodies; D018951:Antigens, CD20; D000939:Epitopes