The use of intracellular single-chain antibody fragments to inhibit specifically the expression of cell surface molecules.
One possible method to inhibit specifically the function of a protein inside a cell is to express an intracellular antibody combining site that can block function or prevent expression of the targeted molecule. In this report the parameters involved in the production and expression of functional, endoplasmic reticulum-retained, single chain Fv antibody fragments (scFv) were investigated. These intracellular scFv constructs were tested for their ability to inhibit specifically the expression of a CHO cell line pretransfected with the relevant cell surface antigen CD2. No scFv was detected in the cell supernatant although functional scFv, as assayed by ELISA, was detected in an NP-40 soluble fraction if an N-linked glycosylation site had been introduced into the antibody construct. This demonstrates that functional antibody combining sites can be produced even though they are not secreted. Inhibition of CD2 was obtained but was not complete and differed between clones. Levels of scFv could be increased by gene amplification but the level of functional binding activity remained constant and no further inhibition of CD2 expression was obtained. Immunofluorescence analysis at the single-cell level of the permeabilised transfected cell lines showed that less than 8% of cells expressed detectable levels of intracellular scFv, indicating selection against cells producing high levels of single-chain antibody. This selection was not seen when comparable single-chain TCR constructs, known to be retained intracellularly, were used. Thus, production of scFv with binding activity is not sufficient for good inhibition of gene expression although introduction of an N-linked glycosylation site is beneficial. The best strategy is probably to screen a panel of scFv constructs and use those that are secreted rather than those that are retained intracellularly.
Greenman
J
J
MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, UK.
Jones
E
E
Wright
M D
MD
Barclay
A N
AN
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000918:Antibody Specificity; D001483:Base Sequence; D001665:Binding Sites; D018801:CD2 Antigens / Q000096:biosynthesis*; D016466:CHO Cells / Q000378:metabolism / Q000502:physiology; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D018801:CD2 Antigens; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments