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Toward selection of internalizing antibodies from phage libraries
B Becerril1, M A Poul, J D Marks
1Department of Pharmaceutical Chemistry, University of California, San Francisco 94110, USA.
Biochemical and Biophysical Research Communications
|March 2, 1999
Summary
This study shows that phage display can directly select internalizing antibodies targeting ErbB2. Optimized formats, like bivalent diabodies, enhance antibody delivery into cells for therapeutic applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Background:
- Internalizing antibodies are crucial for targeted drug and DNA delivery into mammalian cells.
- Traditional methods for identifying these antibodies involve screening hybridomas.
- Phage display offers a potential alternative for direct selection of internalizing antibodies.
Purpose of the Study:
- To evaluate the feasibility of directly selecting internalizing antibodies from phage libraries.
- To identify the most efficient display format for antibody-mediated cellular uptake.
- To investigate the roles of affinity, valency, and display format in phage endocytosis.
Main Methods:
- Utilized a human single-chain variable fragment (scFv) targeting ErbB2 (C6.5).
- Displayed C6.5 scFv monovalently on a phagemid to demonstrate receptor-mediated endocytosis.
- Engineered affinity mutants and dimeric diabodies, displayed on phagemid or phage, to assess endocytosis efficiency.
- Measured phage recovery from cytosol as a function of phage titer and cell preincubation with chloroquine.
Main Results:
- Demonstrated that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis.
- Phage displaying bivalent diabodies or multiple scFv copies showed more efficient endocytosis than monomeric scFv.
- Preincubation with chloroquine increased the recovery of infectious phage.
- Confirmed the possibility of selecting for endocytosable antibodies even at low concentrations within large libraries.
Conclusions:
- Phage display is a viable method for directly selecting internalizing antibodies.
- Antibody valency and display format significantly influence endocytosis efficiency.
- Optimized phage display formats can enhance the delivery of therapeutic payloads into cells.