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

• Published in: Journal of immunological methods 2005-02, Vol.297 (1), p.109-124
• Main Authors: Hamdy, Nayera, Goustin, Anton Scott, Desaulniers, Jean-Paul, Li, Mei, Chow, Christine S., Al-Katib, Ayad
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antigens, CD20 - analysis; Antigens, CD20 - immunology; Antigens, Neoplasm - analysis; Antigens, Neoplasm - immunology; B-lymphocytes; Base Sequence; Biological and medical sciences; CD20; Cloning, Molecular; Erythrocytes - immunology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Glycosylphosphatidylinositols - genetics; Human antibodies; Humans; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Immunoglobulin Variable Region - therapeutic use; Mice; Molecular immunology; Molecular Sequence Data; Neoplasms - therapy; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Rosette Formation; scFv; Sheep; Techniques
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2004.12.003

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 κ regions from a mouse hybridoma (HB-9645) directed against human CD20. In addition to the basic scFv construct (V κ-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.