title: Sheep red blood cells armed with anti-CD20 single-chain variable fragments (scFvs) fused to a glycosylphosphatidylinositol (GPI) anchor: a strategy to target CD20-positive tumor cells.
abstract: Single-chain variable fragment antibodies (scFv) retain antigen specificity and offer advantages over intact antibodies as therapeutic agents. We cloned the cDNA of the V(H) and V(kappa) regions from a mouse hybridoma (HB-9645) directed against human CD20. In addition to the basic scFv construct (V(kappa)-L-V(H)), we genetically engineered a secretory signal, six histidine residues, and a 'Flu' tag to facilitate secretion, purification, and detection. A glycosyl-phosphatidylinositol (GPI) modification signal was added at the C terminus. The GPI-tagged and the non-tagged scFvs were expressed in high yields on the surface of stably transfected insect cells. The CD20-binding properties of purified non-GPI tagged scFv were examined using flow cytometry and immunocytochemistry. The non-GPI-tagged scFv selectively recognizes CD20-positive cells in a concentration-dependent manner. Double-flow cytometry analysis using fresh peripheral blood lymphocytes and WSU-FSCCL cells revealed that our scFv resolves the B-cell population better than the intact antibody. The GPI-tagged scFv was loaded onto the surface of sheep erythrocytes to form rosettes with CD20-positive cells. The genetically engineered anti-CD20 scFv and GPI-tagged derivative have binding specificity for the CD20 antigen. The scFvs described here has potential uses as an in vivo tumor-imaging agent and as a carrier vehicle for targeted delivery of cytocidal agents to CD20-positive cancer cells.
authors.affiliation: Lymphoma Research Laboratory, Department of Internal Medicine, School of Medicine, Wayne State University Detroit, MI 48201, USA.
authors.forename: Nayera
authors.initials: N
authors.lastname: Hamdy
authors.forename: Anton Scott
authors.initials: AS
authors.lastname: Goustin
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Desaulniers
authors.forename: Mei
authors.initials: M
authors.lastname: Li
authors.forename: Christine S
authors.initials: CS
authors.lastname: Chow
authors.forename: Ayad
authors.initials: A
authors.lastname: Al-Katib
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018951:Antigens, CD20 / Q000032:analysis / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000032:analysis / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000276:immunology; D017261:Glycosylphosphatidylinositols / Q000235:genetics*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D051379:Mice; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000628:therapy*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012397:Rosette Formation; D012756:Sheep
chemical_list: D018951:Antigens, CD20; D000951:Antigens, Neoplasm; D017261:Glycosylphosphatidylinositols; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins