title: Design, expression and characterization of a single chain anti-CD20 antibody; a germline humanized antibody derived from Rituximab.
abstract: CD20 is a B cell lineage specific surface antigen involved in various B cell malignancies. So far, several murine and chimeric antibodies have been produced against this antigen among which Rituximab is a commercially approved antibody widely used in treatment of cancers associated with CD20 overexpression. The current study reports the production and characterization of a humanized single chain version of Rituximab through CDR grafting method. For either heavy or light chain variable domains, a human antibody with the highest sequence homology to Rituximab was selected from human germline sequences and used as framework donors. Vernier zone residues in framework regions were replaced with those of Rituximab to retain the antigen binding affinity of parental antibody. The reactivity of humanized single chain antibody with CD20 was examined by ELISA and dot blot assays. The ability of antibody to suppress the growth of CD20 overexpressing Raji cells was tested by MTT assay. Analysis of reactivity with CD20 antigen revealed that the humanized single chain antibody reacted to the target antigen with high affinity. Proliferation inhibition assay showed that humanized scFv could suppress the proliferation of Raji cells efficiently in a dose-dependent manner. This successful production of a humanized scFv with the ability to inhibit growth of CD20-expressing cancer cell may provide a promising alternative strategy for CD20 targeted therapy.
authors.affiliation: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Vahideh
authors.initials: V
authors.lastname: Ahmadzadeh
authors.affiliation: Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Electronic address: farajnias@tbzmed.ac.ir.
authors.forename: Safar
authors.initials: S
authors.lastname: Farajnia
authors.affiliation: Department of Biology, University of Tabriz, Tabriz, Iran.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Hosseinpour Feizi
authors.affiliation: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Ramazan Ali
authors.initials: RA
authors.lastname: Khavarinejad
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry / Q000235:genetics; D018951:Antigens, CD20 / Q000276:immunology*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000188:drug therapy / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D000069283:Rituximab; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D000069283:Rituximab
keywords: Anti-CD20; Germ line humanization; Single chain antibody fragment