In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library
| Autor: | Michael A. Bookman, Robert Schier, John E. McCartney, E J Wolf, L L Houston, Gerald Apell, C Wong, Louis M. Weiner, James S. Huston, James D. Marks |
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| Rok vydání: | 1995 |
| Předmět: |
Phage display
Receptor ErbB-2 medicine.medical_treatment Immunology Molecular Sequence Data Immunoglobulin Variable Region Enzyme-Linked Immunosorbent Assay CHO Cells Mice SCID Mice Antigen In vivo Peptide Library Cricetinae medicine Animals Humans Amino Acid Sequence Peptide library Immunoglobulin Fragments biology Chemistry Molecular biology In vitro Recombinant Proteins Radioimmunotherapy biology.protein Hybridoma technology Immunoglobulin Light Chains Antibody Immunoglobulin Heavy Chains |
| Zdroj: | Immunotechnology : an international journal of immunological engineering. 1(1) |
| ISSN: | 1380-2933 |
| Popis: | 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 K d of 1.6 × 10 −8 M and to c-erbB-2 on SK-OV-3 cells with a K d of 2.0 × 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 125 I-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 nonimmune 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. |
| Databáze: | OpenAIRE |
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