Targeting vectors for intracellular immunisation.

We define intracellular immunization as the inhibition or inactivation of the function of a molecule by the ectopic intracellular expression of antibody binding domains which recognise the molecule. Such recombinant antibodies can be directed to different compartments of eukaryotic cells by means of previously defined targeting signals, thus permiting the study of any molecule in any cellular compartment for which an antibody is available. For this purpose, we have created a set of vectors based on the VHExpress vector described [Persic, L., Roberts, A., Wilton, J., Cattaneo, A., Bradbury, A. and Hoogenboom, H.R. (1997) An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187, 000-000], which has been modified to express scFvs (single chain fragments) linked to specific targeting signals. These permit the localisation of scFvs to different intracellular compartments: the endoplasmic reticulum (scFvE-er), the nucleus (scFvE-nuclear), the mitochondria (scFvE-mit), the cytoplasm (scFvE-cyto), and as secreted proteins (scFvE-sec). The function of these vectors has been assessed by the immunofluorescence of COS cells transiently transfected with constructs containing the alphaD11 scFv.

An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries.

Phage display is now an established method to select antibody fragments specific for a wide range of diverse antigens. In particular, isolation of human monoclonal antibodies has become a reality and for most purposes bacterial expression of the selected recombinant antibody fragments is sufficient. However, there are some cases where the expression of complete human immunoglobulin in mammalian cells is, if not essential, at least desirable. For this reason we have designed and constructed a set of mammalian expression vectors which permit facile and rapid cloning of antibody genes for both transient and stable expression in mammalian cells. Immunoglobulin genes may be cloned into these expression vectors as V regions or as Fabs for expression as either complete antibodies or as Fab fragments, using restriction sites which are rare in human V genes. All the important elements in the vectors--promoter, leader sequence, constant domains and selectable markers--are flanked by unique restriction sites, allowing simple substitution of elements. The vectors have been evaluated using the variable regions from the neutralizing anti-nerve growth factor (NGF) antibody, alphaD11, and the V regions from 2E10, a scFv selected from a scFv phagemid library.

Single-chain variable fragments selected on the 57-76 p21Ras neutralising epitope from phage antibody libraries recognise the parental protein.

Phage antibodies have been widely prospected as an alternative to the use of monoclonal antibodies prepared by traditional means. Many monoclonal antibodies prepared against peptides are able to recognise the native proteins from which they were derived. Here we show that the same is also true for phage antibodies. We have selected a number of single-chain variable fragments (scFv) from a large phage scFv library against a peptide from the switch region II of p21Ras. This peptide is known to reside in a mobile area of the native protein and is the epitope of a well characterised monoclonal antibody. Selected scFvs were able to recognise native p21Ras in both ELISA and Western blots, indicating that peptides are also likely to be very useful in selecting from phage antibody libraries.

Effect of saccharin on the meiotic division of Saccharomyces cerevisiae.

The effect of saccharin on the occurrence of meiotic diploid and disomic products in Saccharomyces cerevisiae was investigated. It was found that this substance inhibits the sporulation process in a dose-dependent manner, is ineffectual on recombination frequency, and slightly increases the occurrence of diploid and disomic meiotic products. It is demonstrated that the formation of diploid meiotic products is a consequence of random nuclear fusions at the end of the meiotic process or of endomitosis preceding meiosis.

Use of living columns to select specific phage antibodies.

Here we demonstrate that it is possible to confront two recombinant microorganisms in order to select one using the other. We have shown that an epitope derived from p21ras expressed within the outer membrane protein, LamB, can be recognized both by the monoclonal antibody Y13-259, as well as the single chain Fv fragment derived from it. This specificity, which is maintained when the Y13-259 single chain Fv is expressed as a fusion protein with the phage fd gene 3 protein, has allowed us to use the living column of LamB-ras to purify Y13-259 phage from a background of non-binding phage, even at dilutions as high as 10 phage in 10(10) irrelevant phage.