Anti-idiotypic antibody as the surrogate antigen for cloning scFv and its fusion proteins.
Single-chain variable fragment (ScFv) is a versatile building block for novel targeting constructs. However, a reliable screening and binding assay is often the limiting step for antigens that are difficult to clone or purify. Anti-idiotypic antibodies may be useful as surrogate antigens for cloning scFv and their fusion proteins. 8H9 is a murine IgG(1) monoclonal antibody (MAb) specific for a novel antigen expressed on the cell surface of a wide spectrum of human solid tumors, but not in normal tissues. Rat anti-8H9-idiotypic hybridomas (clones 2E9, 1E12, and 1F11) were produced by somatic cell fusion between rat lymphocytes and mouse SP2/0 myeloma. In direct binding assays enzyme-linked immunosorbant assay--(ELISA)--they were specific for the 8H9 idiotope. Using 2E9 as the surrogate antigen, 8H9-scFv was cloned from hybridoma cDNA by phage display. 8H9scFv was then fused to human-gamma1-CH2-CH3 cDNA for transduction into CHO and NSO cells. High expressors of mouse scFv-human Fc chimeric antibody were selected. The secreted homodimer reacted specifically with antigen-positive tumor cells by ELISA and by flow cytometry, inhibitable by the anti-idiotypic antibody. The reduced size resulted in a shorter half-life in vivo, while achieving comparable tumor to nontumor ratio as the native antibody 8H9. However, its in vitro activity in antibody-dependent cell-mediated cytotoxicity was modest.
Cheung
Nai-Kong V
NK
Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. cheungn@mskcc.org
Guo
Hong-Fen
HF
Modak
Shakeel
S
Cheung
Irene Y
IY
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D003001:Cloning, Molecular; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology
D000888:Antibodies, Anti-Idiotypic; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins