251836 A Platform for Antibody Engineering Against Membrane Protein Targets Using Detergent-Solubilized Cell Lysates As Antigen Sources

Monday, October 29, 2012: 12:30 PM
Fayette (Westin )
Yong Ku Cho1, Ben J. Tillotson1, Inigo de Larrinoa2 and Eric V. Shusta1, (1)Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, (2)Applied Chemistry, University of the Basque Country, San Sebastian, Spain

Antibodies that target plasma membrane proteins are often attractive reagents for research and therapeutic purposes.  However, membrane proteins are challenging to work with in terms of antibody selection, engineering, and antigen identification as a result of their insolubility in aqueous solutions.  We have therefore developed a platform for antibody engineering using detergent-solubilized cell lysates as antigen sources.  Such approaches are compatible with membrane protein targets, subcellular selections, and the rapid assessment of antibody specificity. To demonstrate, we screened a nonimmune human single-chain antibody library directly against detergent-solubilized cell lysates to identify antibodies binding plasma membrane proteins.  Because they were selected using nondenaturing detergents, these antibodies retained functional binding capacity under physiological conditions.  Moreover, when combined with yeast display immunoprecipitation procedures, antigen analysis was facilitated.  For example, the antibody-displaying yeast were used directly to immunoprecipitate cognate membrane protein ligands from cell lysates and this immunoprecipitation product was used to explore antigen size and diversity as well as for de novo antigen identification through tandem mass spectrometric protein sequencing. Finally, we show that detergent-solubilized mammalian cells constitute a crude antigen solution that can be used for antibody affinity maturation. Yeast surface display was employed as a platform to screen a mutagenic library of anti-human transferrin receptor (hTfR) antibodies using detergent-solubilized cell lysates.  An off-rate selection yielded scFv having 2-8 fold improved binding affinity to hTfR. Taken together, the combined cell lysate and yeast antibody display platform facilitates antibody engineering against membrane proteins at many levels and could prove to be a powerful platform as the interest in membrane proteins continues to increase.

Extended Abstract: File Not Uploaded