Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments

Abstract

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the antip53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 7/7 human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 7/7 human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant de®cient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

“…Moreover, the transcriptional transactivation function of the common mutant R273H was restored by microinjection of PAb421 antibody or PAb421-derived Fv fragments in tumor cells (Abarzua et al, 1995;Caron de Fromentel et al, 1999). In addition, several monoclonal antibodies that recognize N-terminal epitopes can partially rescue the DNA binding of p53 mutants (Cohen et al, 1999).…”

Strategies for manipulating the p53 pathway in the treatment of human cancer

“…The yellow arrow represents the shift in equilibrium promoted by mutation from the folded active tetramer (light-grey squares) to the folded latent tetramer (dark-grey ellipses). The allosteric mutant class of p53 can be activated in vivo by agents that target the C-terminal negative regulatory domain [52,53,261,262]. (C) Thermodynamic stability.…”

“…One class of activating agents (1) including a C-terminal peptide (C-t peptide) or a monoclonal antibody (PAb421 MAb), promotes the conversion of p53 tetramers from a latent (dark-grey ellipses) into an activated (lilac squares) state by neutralizing its C-terminal negative regulatory domain. This increases the specific activity of p53 as a sequence-specific DNA-binding protein and stimulates p53-dependent transcription and apoptosis [45,51,53,150,261,262]. (ii) Inhibiting MDM2 binding to p53.…”

“…Increased phosphorylation at Ser$*# of endogenous human p53 protein in cells occurs following UV-C and X-irradiation damage, with enhanced steady-state phosphorylation correlating with enhanced p53-dependent transcription [49,50]. In addition, microinjection or intracellular synthesis of an antibody (PAb421) that binds to the C-terminal negative regulatory domain near the phosphorylation sites ( Figures 2 and 4) can activate p53-dependent gene expression in i o [45,[51][52][53], suggesting that Cterminal modification can be a rate-limiting step in stimulating p53 function in i o (Figure 4). …”

“…Similarly, small peptides that can activate the latent DNA-binding function of p53 in itro can also activate p53 function in i o when coupled to carrier protein or ligand [52,261,262]. The intracellular synthesis of a single-chain PAb421 antibody expression cassette can also activate allosteric p53 mutants, but not structural p53 mutants in i o [53], highlighting the biochemical distinctions that exist between p53 mutant proteins and the need to define the class of mutant p53 existing within cancers if activating drugs are in fact developed and used. These studies have predicted the possibility that cellular enzymes which modify the C-terminal domain may exist, and now a variety of enzymes that target the C-terminal domain are thought to play a stimulatory role in the p53 pathway after cell injury (Figure 2).…”