BACKGROUND: Antibody-based reagents have failed to live up to their anticipated role as highly specific targeting agents for cancer therapy. Targeting with human single-chain Fv (sFv) molecules may overcome some of the limitations of murine IgG, but are difficult to produce with conventional hybridoma technology. Alternatively, phage display of antibody gene repertoires can be used to produce human sFv. OBJECTIVES: To isolate and characterize human single chain Fvs which bind to c-erbB-2, an oncogene product overexpressed by 30-50% of breast carcinomas and other adenocarcinomas. STUDY DESIGN: A non-immune human single-chain Fv phage antibody library was selected on human c-erbB extracellular domain and sFv characterized with respect to affinity, binding kinetics, and in vivo pharmacokinetics in tumor-bearing scid mice. RESULTS: A human single-chain Fv (C6.5) was isolated which binds specifically to c-erbB-2. C6.5 is entirely human in sequence, expresses at high level as native protein in E. coli, and is easily purified in high yield in two steps. C6.5 binds to immobilized c-erbB-2 extracellular domain with a Kd of 1.6 x 10(-8) M and to c-erbB-2 on SK-OV-3 cells with a Kd of 2.0 x 10(-8) M, an affinity that is similar to sFv produced against the same antigen from hybridomas. Biodistribution studies demonstrate 1.47% injected dose/g tumor 24 h after injection of 125I-C6.5 into scid mice bearing SK-OV-3 tumors. Tumor:normal organ ratios range from 8.9:1 for kidney to 283:1 for muscle. CONCLUSIONS: These results are the first in vivo biodistribution studies using an sFv isolated from a non-immune human repertoire and confirm the specificity of sFv produced in this manner. The use of phage display to produce C6.5 mutants with higher affinity and slower k(off) would permit rigorous evaluation of the role of antibody affinity and binding kinetics in tumor targeting, and could result in the production of a therapeutically useful targeting protein for radioimmunotherapy and other applications.
BACKGROUND: Antibody-based reagents have failed to live up to their anticipated role as highly specific targeting agents for cancer therapy. Targeting with human single-chain Fv (sFv) molecules may overcome some of the limitations of murine IgG, but are difficult to produce with conventional hybridoma technology. Alternatively, phage display of antibody gene repertoires can be used to produce human sFv. OBJECTIVES: To isolate and characterize human single chain Fvs which bind to c-erbB-2, an oncogene product overexpressed by 30-50% of breast carcinomas and other adenocarcinomas. STUDY DESIGN: A non-immune human single-chain Fv phage antibody library was selected on human c-erbB extracellular domain and sFv characterized with respect to affinity, binding kinetics, and in vivo pharmacokinetics in tumor-bearing scid mice. RESULTS: A human single-chain Fv (C6.5) was isolated which binds specifically to c-erbB-2. C6.5 is entirely human in sequence, expresses at high level as native protein in E. coli, and is easily purified in high yield in two steps. C6.5 binds to immobilized c-erbB-2 extracellular domain with a Kd of 1.6 x 10(-8) M and to c-erbB-2 on SK-OV-3 cells with a Kd of 2.0 x 10(-8) M, an affinity that is similar to sFv produced against the same antigen from hybridomas. Biodistribution studies demonstrate 1.47% injected dose/g tumor 24 h after injection of 125I-C6.5 into scid mice bearing SK-OV-3 tumors. Tumor:normal organ ratios range from 8.9:1 for kidney to 283:1 for muscle. CONCLUSIONS: These results are the first in vivo biodistribution studies using an sFv isolated from a non-immune human repertoire and confirm the specificity of sFv produced in this manner. The use of phage display to produce C6.5 mutants with higher affinity and slower k(off) would permit rigorous evaluation of the role of antibody affinity and binding kinetics in tumor targeting, and could result in the production of a therapeutically useful targeting protein for radioimmunotherapy and other applications.
Authors: Tracy R Daniels; Richard K Leuchter; Rafaela Quintero; Gustavo Helguera; José A Rodríguez; Otoniel Martínez-Maza; Birgit C Schultes; Christopher F Nicodemus; Manuel L Penichet Journal: Cancer Immunol Immunother Date: 2011-11-30 Impact factor: 6.968
Authors: Yu Zhou; Daryl C Drummond; Hao Zou; Mark E Hayes; Gregory P Adams; Dmitri B Kirpotin; James D Marks Journal: J Mol Biol Date: 2007-05-10 Impact factor: 5.469
Authors: Yu Zhou; Hao Zou; Christina Yau; Lequn Zhao; Steven C Hall; Daryl C Drummond; Shauna Farr-Jones; John W Park; Christopher C Benz; James D Marks Journal: Protein Eng Des Sel Date: 2018-01-01 Impact factor: 1.650
Authors: Keyu Li; Kirstin A Zettlitz; Julia Lipianskaya; Yu Zhou; James D Marks; Parag Mallick; Robert E Reiter; Anna M Wu Journal: Protein Eng Des Sel Date: 2015-05-19 Impact factor: 1.650
Authors: Mark Throsby; Cecile Geuijen; Jaap Goudsmit; Arjen Q Bakker; Jehanara Korimbocus; R Arjen Kramer; Marieke Clijsters-van der Horst; Maureen de Jong; Mandy Jongeneelen; Sandra Thijsse; Renate Smit; Therese J Visser; Nora Bijl; Wilfred E Marissen; Mark Loeb; David J Kelvin; Wolfgang Preiser; Jan ter Meulen; John de Kruif Journal: J Virol Date: 2006-07 Impact factor: 5.103
Authors: Ann-Charlott Steffen; Anna Orlova; Maria Wikman; Fredrik Y Nilsson; Stefan Ståhl; Gregory P Adams; Vladimir Tolmachev; Jörgen Carlsson Journal: Eur J Nucl Med Mol Imaging Date: 2006-03-15 Impact factor: 9.236