Transfected cell microarrays for the expression of membrane-displayed single-chain antibodies.
The expression of recombinant antibody fragments on the surface of mammalian cells has recently emerged as a therapeutic strategy, particularly in the treatment of a number of cancers. Screening technologies that allow for the facile characterization of fragments expressed on the cell surface would hasten the identification and isolation of reagents to be used as therapeutics. In this report, we describe a cellular microarray-based platform for the comparative functional analysis of single-chain antibodies (scFvs) expressed on the plasma membrane of mammalian cells. Using the anti-fluorescein monoclonal antibody 4-4-20 as a model system, the native binding site and three mutants were expressed as scFvs on the membrane of HEK 293T/17 cells in a microarray format. Collectively, the equilibrium dissociation constants of the soluble forms of the wild-type scFv and the three mutants spanned nearly 3 orders of magnitude. Expression of the scFvs on the surface of mammalian cells was achieved by the deposition of plasmid DNAs in micrometer-sized spots onto the surface of a glass microscope slide. The addition of cells to the printed array resulted in the expression of the scFvs in clusters of cells in spatially discrete locations. Ligand binding assays performed with a fluorescein-bovine serum albumin conjugate demonstrated the ability of the transfected cell microarray to differentiate the relative binding affinities of the expressed scFvs. Further, the apparent affinities of the membrane-displayed scFvs were within 10-fold of those reported for the soluble forms of the scFvs. The assays described herein demonstrate the potential for cellular microarrays to be used for the high-throughput screening of potential therapeutic reagents. More generally, our work details the utility of transfected cell microarrays in mediating the functional characterization of expressed membrane receptor proteins.
Delehanty
James B
JB
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, D.C. 20375-5348, USA. jbd@cbmse.nrl.navy.mil
Shaffer
Kara M
KM
Lin
Baochuan
B
D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D019793:Fluorescein; D005786:Gene Expression Regulation; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009369:Neoplasms / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D014162:Transfection
D000906:Antibodies; D000970:Antineoplastic Agents; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; C055966:immunoglobulin Fv; D019793:Fluorescein