Characterization of scFv-Ig constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths
| Autor: | Shan D, Oliver Press, Tt, Tsu, Ms, Hayden, Ja, Ledbetter |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
B-Lymphocytes
Base Sequence Blotting Western Molecular Sequence Data Immunoglobulin Variable Region Antibodies Monoclonal Apoptosis Enzyme-Linked Immunosorbent Assay Antigens CD20 Flow Cytometry Tumor Cells Cultured Humans Amino Acid Sequence Binding Sites Antibody Immunoglobulin Heavy Chains Peptides Chromatography High Pressure Liquid DNA Primers |
| Zdroj: | Europe PubMed Central |
| ISSN: | 0022-1767 |
| Popis: | The heavy (VH) and light (VL) chain variable regions of the murine anti-human CD20 mAb 1F5 were cloned, and four single-chain Ab (scFv) molecules were constructed using linker peptides of variable lengths to join the VH and VL domains. Three constructs were engineered using linker peptides of 15, 10, and 5 aa residues consisting of (GGGGS)3, (GGGGS)2, and (GGGGS)1 sequences, respectively, whereas the fourth was prepared by joining the VH and VL domains directly. Each construct was fused to a derivative of human IgG1 (hinge plus CH2 plus CH3) to facilitate purification using staphylococcal protein A. The aggregation and CD20 binding properties of these four 1F5 scFv-Ig derivatives produced were investigated. Both size-exclusion HPLC column analysis and Western blots of proteins subjected to nonreducing SDS-PAGE suggested that all four 1F5 scFv-Ig were monomeric with m.w. of approximately 55 kDa. The CD20 binding properties of the four 1F5 scFv-Ig were studied by ELISA and flow cytometry. The 1F5 scFv-Ig with the 5-aa linker (GS1) demonstrated significantly superior binding to CD20-expressing target cells, compared with the other scFv-Ig constructs. Scatchard analysis of the radiolabeled monovalent GS1 scFv-Ig revealed a binding avidity of 1.35 x 108 M-1 compared with an avidity of 7.56 x 108 M-1 for the native bivalent 1F5 Ab. These findings suggest that the GS1 scFv-Ig with a short linker peptide of approximately 5 aa is the best of the engineered constructs for future studies. |
| Databáze: | OpenAIRE |
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