The following references are referenced in the specification and may be relevant to understanding the invention:    1. Gamble, J. and Milner, J. (1988). Evidence that immunological variants of p53 represent alternative protein conformations. Virology, 162, 452-458.    2. Harlow, E., Crawford, L. V., Pim, D. C. and Williamson, N. H. (1981). Monoclonal antibodies specific for simian virus 40 tumor antigens. J. Virol, 39, 861-869.    3. Yewdell, J. W., Gannon, J. V. and Lane, D. P. (1986). Monoclonal antibody analysis of p53 expression in normal and transformed cells. J. Virol. 59, 444-452.    4. Milner, J., Cook, A. and Sheldon, M. (1987). A new anti-p53 monoclonal antibody, previously reported to be directed against the large T antigen of simian virus-40. Oncogene, 1, 453-455.    5. Gannon, J. V., Greaves, R., Iggo, R. and Lane, D. P. (1990). Activating mutations in p53 produce common conformation effects. A monoclonal antibody specific for the mutant form. EMBO J. 9, 1595-1602.    6. Marasco, W. A. (1995) Intracellular antibodies (intrabodies) as research reagents and therapeutic molecules for gene therapy. Immunotechnology, 1, 1-19.    7. Stephen. C. W. and Lane, D. P. (1992). Mutant conformation of p53 precise epitope mapping using a filamentous phage epitope library. J Mol Biol. 225, 577-583.    8. Jannot. C. B. and Hynes, N. E. (1997). Biochem. Biophys. Res. Commun., 230, 242-246.    9. Cohen, P. A., Mani, J. C. and Lane, D. P. (1998). Characterization of a new intrabody directed against the N-terminal region of human p53. Oncogene, 17, 2445-2456.
The p53 gene encodes a protein which is 53 kD in size (hence the name). The p53 protein is situated in the nexus of intermingled pathways controlling cell proliferation, cell survival and differentiation. It can sense and integrate various external and internal stimuli, such as DNA damage, hypoxia, oxidative stress, deregulated oncogene expression and ribonucleotide depletion. In response to these stimuli, it may trigger cell cycle arrest, apoptosis, senescence, differentiation or antiangiogenesis. It appears that the p53 protein is involved in regulation of critical growth controlling checkpoints. It is capable of exercising a tumor suppressor function by preventing cells, that are in unfavorable environmental conditions or are carrying damaged DNA, from entering a cell cycle.
Mutations within the p53 gene have been found in more than 50% of all human cancers, rendering it the most frequently mutated single gene in human cancer known so far. Most of the mutations in the p53 gene discovered in cancers are of a missense type. They cause not just abrogation of the tumor suppressor function of wild-type p53, but often actively contribute to the tumor transformation function of mutant p53. Inactivation of p53 is one of the most common molecular events in cancer development.
As mentioned above, the p53 protein, which is both a regulator for cell proliferation and a suppressor of tumor development, can prevent the development of cancer by blocking the division of cells which have sustained DNA damage, or by triggering apoptosis. It has been proposed that the effect of mutant p53 on tumor progression is due to a dominant negative interaction of the wild type and mutant proteins. Thus, wild-type and mutant p53 proteins are respectively capable of contrasting suppressor and promoter effects on tumor development.
The ability of p53 to act both as a suppressor and a promoter of tumor development may reflect the ability of the protein to adopt different conformations. Mitogenic stimulation of primary T cells induces a change in the immunoreactivity of p53 and this may be due to a change in the tertiary structure of the p53 protein. To ascertain whether the ‘mutant’ conformation of wild-type p53 has physiological relevance, attempts have been made to associate it with a biochemical activity. Conformation-specific monoclonal antibodies, previously shown to discriminate between wild-type and mutant p53 proteins, have been used to demonstrate structural changes in wild-type p53 following sequence-specific binding to DNA (1). These studies suggested that wild-type p53 can physiologically adopt distinct conformations, which determine its DNA binding activity. Mutations that render p53 oncogenic may lock p53 into one of the few conformational states it physiologically adopts, rather than distort its tertiary structure.
Two important cellular factors appear necessary for this conversion: (1) binding of an ‘activating’ polypeptide that causes neutralization of the C-terminal negative regulatory domain, and (2) a highly reduced environment which can maintain p53 in an activated state. Given the clear association between the DNA binding activity and tumor suppressor functions of p53, these results imply that in many tumor cells there are high levels of mutant p53 that can potentially be activated to restore significant wild type function.
A variety of monoclonal antibodies have been prepared against various epitopes of p53.
A panel of anti-p53 mouse monoclonal antibodies has been used to characterize the immunoreactivities of the native p53-Ala35 and the mutant p53-Val 35 translated under various conditions. Two monoclonal antibodies, PAb421 and PAb248, were able to recognize discrete denaturation-stable epitopes on the p53-polypeptide (2,3). These antibodies immunoprecipitated both p53-Ala35 and p53-Val 35 translated at 30° C. and 37° C., and were also able to immunoprecipitate p53 from a variety of murine cell lines (4).
Three additional monoclonal antibodies, PAb246, PAb 1620 and PAb240, were found to detect conformational changes in the p53 protein (5). Molecules in the mutant conformation are distinguished from wild-type molecules, inter alia, by the appearance of a new, normally cryptic epitope recognized by Pab240. This epitope was localized to residues 213 to 217 of the p53 protein and has the sequence RHSVV, preceded by F in human and mouse p53 (7). More than 90% of mutations found in p53 produce a conformational change in the p53 protein which results in the exposure of this epitope, which is otherwise hidden in the hydrophobic core of the molecule. This epitope will be referred to hereinafter as the common mutant epitope of p53.
Recent advances in antibody engineering have allowed the genes encoding antibodies to be manipulated, so that antigen binding molecules can be expressed within mammalian cells in a controlled way (6). Application of gene technologies to antibody engineering has enabled the synthesis of single-chain fragment variable (scFv) antibodies that combine within a single polypeptide chain the light and heavy chain variable domains of an antibody molecule covalently joined by a predesigned peptide linker. The resultant scFv gene can be expressed in bacterial expression systems such as E. coli. Bundled in the “gene display package” single-chain antibodies displayed at the surface of filamentous phages of the M13 family provided the possibility to create antibody libraries both from various living sources and products of diversification of a single scFv molecule. Antibodies with the desired specificity can be isolated from such libraries employing effective selection techniques (biopanning) in which the antigen is immobilized on a solid support.
The ability to create scFv antibodies, when combined with their stable expression in precise intracellular locations in mammalian cells, has enabled the creation of a powerful new family of antibody molecules for basic research or gene therapy. These intracellular antibodies (intrabodies) can be used to modulate cellular physiology and metabolism through a variety of mechanisms, including the blocking, stabilizing or mimicking of protein-protein interactions, by altering enzyme function, or by diverting proteins from their usual intracellular compartments. Intrabodies can be directed to the relevant cellular compartments by modifying the genes that encode them to specify N- or C-terminal polypeptide extensions for providing intracellular-trafficking signals. This approach has been described for a number of different antigens, including several HIV proteins.
Several scFv antibodies against p53 have been previously described. The scFv-421 antibody recognizes a C-terminal epitope of the protein (8). When expressed in vitro in the cytoplasm or in the nucleus of COS-1 cells, it was found to be non-functional and prone to rapid degradation. An important determinant of correct antibody folding is the formation of intra-chain disulfide bonds in the variable regions; possibly the reducing environment of the cytosol may lead to a decrease in the stability of the scFv (9). Nevertheless, some scFv antibodies have been expressed in the cytoplasm and shown to have biological effects, indicating that other features, such as the primary sequence of the antibody and/or its specific cellular location may be important for their proper function.
The scFv DO-i antibody was found to recognize an N-terminal epitope of human p53 (9). The DO-1 scFv was targeted to the cytoplasm and to the nucleus of mammalian cells. Interestingly, insertion of the CK domain into scFv to create a scFvCK fusion protein, led to a dramatic increase in the level of intracellular expression. However, in other studies in which CK fusions to scFv were made, the effects on stability and expression were much less marked. It is clear that each scFv is a particular and individual case (9).