Addition of TAT protein transduction domain and GrpE to human p53 provides soluble fusion proteins that can be transduced into dendritic cells and elicit p53-specific T-cell responses in HLA-A*0201 transgenic mice

Abstract

The protein p53 has been shown to be an efficient tumour antigen in both murine and human cancer vaccine studies and cancer vaccines targeting p53 based on major histocompatibility complex (MHC) class I binding p53-derived peptides that induce cytotoxic T lymphocytes (CTLs) without p53-specific CD4(+) T-cell help have been tested by several research groups including ours. To obtain such CD4(+) T-cell help and cover a broader repertoire of MHC haplotypes we have previously attempted to produce recombinant human p53 for vaccination purposes. However, attempts to refold a hexahis-tagged p53 protein in our laboratory were unsuccessful. Here, we show that fusion of an 11-amino-acid region of the human immunodeficiency virus TAT protein transduction domain (PTD) to human p53 increases the solubility of the otherwise insoluble p53 protein and this rTAT-p53 protein can be transduced into human monocyte-derived dendritic cells (DCs). The induction of a p53-specific HLA-A*0201 immune response was tested in HLA-A*0201/K(b) transgenic mice after immunization with rTAT-p53-transduced bone-marrow-derived DCs. In these mice, p53-specific CD4(+) and CD8(+) T-cell proliferation was observed and immunization resulted in the induction of HLA-A*0201-restricted CTLs specific for two human p53-derived HLA-A*0201-binding peptides, p53(65-73) and p53(149-157). Addition of GrpE to generate rTAT-GrpE-p53 led to a further increase in protein solubility and to a small increase in DC maturation but did not increase the observed p53-specific T-cell responses. The use of rTAT-p53 in ongoing clinical protocols should be applicable and offers advantages to current strategies omitting the use of HLA-typed patients.

Figure 1

The p53-containing fusion proteins. Schematic representation of bacterial recombinant proteins. Top panel: rTAT-p53, MW 46 150; pI, 8·45. Bottom panel: rTAT-GrpE-p53, MW, 68 232; pI, 5·91.

Figure 2

SDS–PAGE analysis of p53 fusion proteins. rTAT-GrpE-p53 (a) and rTAT-p53 (b) purified urea-denatured proteins before refolding. Large gel lanes C and D show dilutions of rTAT-GrpE-p53 (lane C) and rTAT-p53 (lane D).

Figure 3

Protein transduction of p53 fusion proteins. Human monocyte-derived DCs were either unpulsed (a) or pulsed with p53 proteins rTAT-GrpE-p53 or rTAT-p53 (b) and (c) and control proteins including rTAT-GrpE and TAT (d) and (e) or a buffer control (f) for 18 hr as indicated. Cell pellets were then incubated with anti-human p53 antibody to block p53 sites from externally deposited protein. Hereafter, cells were stained for intracellular flow cytometry with a PE-conjugated anti-human p53 antibody from the same clone as the blocking antibody or with a relevant isotype control.

Figure 4

Up-regulation of DC activation markers after transduction with rTAT-GrpE-p53. Human monocyte-derived DCs were either unpulsed or pulsed with rTAT-GrpE-p53 for 18 hr as indicated, labelled with antibodies against CD11c (FL1) and activation markers (FL2) or isotype controls and analysed by flow cytometry.

Figure 5

The p53-specific T-cell proliferation in HLA-A*0201/K^b transgenic mice vaccinated with p53 fusion protein-transduced DCs. (a) Mice received either no vaccination, or a vaccination with bone-marrow-derived DCs pulsed with rTAT-p53, rTAT-GrpE-p53 or unpulsed DCs intradermally at the base of the tail. Twenty days after vaccination spleen cells were isolated and cocultured with bone-marrow-derived DCs pulsed with p53 proteins as indicated by the different coloured histograms. Alternatively, the coculture was performed in the presence of CD4- or CD8-blocking antibodies (b).

Figure 6

Induction of p53-specific CTLs recognizing HLA-A*0201-binding peptides p53₆₅₋₇₃ and p53_149−157. Mice received either no vaccination, or a vaccination with bone-marrow-derived DCs pulsed with rTAT-p53, rTAT-GrpE-p53 or unpulsed DCs intradermally at the base of the tail. Twenty days after vaccination, spleen cells from four immunized mice were harvested and cultured together with 10 μm of the indicated peptide and 100 IU/ml IL-2 on the second day. After 9 days, cells were harvested and set up in the ELISPOT assay in triplicates in the presence or absence of the relevant peptide.