Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR

Abstract

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the alpha- and beta-chain of a TCR from a highly avid murine CTL clone that recognized the human p53(264-272) epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.

FIGURE 1

Functional analysis of p53 TCR. A, RNA electroporation of stimulated PBL. PBLs stimulated with OKT3 Ab plus IL-2 were electroporated with in vitro-transcribed RNA at 2 μg/1 × 10⁶ cells. Twenty hours after electroporation, murine TCR expression was determined by FACS analysis for PBLs electroporated with RNA encoding p53 TCR that were stained with anti-murine TCR (1), with isotype control (2), and for non-electroporated cells stained with anti-murine TCR (3). B, PBLs were elec-troporated with p53 TCR RNA and cocultured with peptide-pulsed T2 cells. Peptides used were HLA-A2-restricted p53_{264 –272} and NY-ESO-1_157–165V. C, Schematic representation of the retroviral vector encoding the p53 TCR. LTR, Long terminal repeat.

FIGURE 2

Efficiency of the retroviral transduction. Human PBLs were transduced with the retroviral vector encoding the p53 TCR. A–D, Expression was assessed by FACS 48 h after transduction. The percentage of PBLs that express the mouse TCR (mTCR) of interest are as shown.

FIGURE 3

Recognition of peptide-pulsed and p53-transfected cells. A, Human PBLs expressing the p53 TCR were cocultured for 16 h with T2 cells pulsed with different concentrations of p53-specific peptide (p53_264–272). The concentration of IFN-γ secreted in the medium was measured using an ELISA procedure. IFN-γ secretion in cultures with T2 pulsed with control peptides (gp_100–209, gp_100–280, MART-1_26–35, p53_149–157, and HBVc) was ≤480 pg/ml (data not shown). B, Human PBLs expressing the p53 TCR were cocultured for 16 h with CosA2 cells that were transfected with different p53 (both wt and mutant) plasmid expression vectors. The concentration of IFN-γ secreted in the medium was measured using ELISA.

FIGURE 4

Recognition of human tumor lines. Human PBLs expressing the p53 TCR were cocultured for 16 h with the indicated tumor cell lines. The concentration of IFN-γ secreted in the medium was measured using an ELISA.

FIGURE 5

Tumor cell-mediated IL-2 synthesis. Human PBLs expressing the p53 TCR were cocultured for 16 h with the indicated tumor cell lines. The concentration of IL-2 secreted in the medium was measured using an ELISA.

FIGURE 6

In vitro proliferation of TCR-engineered PBLs. Human PBLs expressing the p53 TCR were labeled with CFSE and cocultured with tumor cell lines. Ag-specific proliferation was measured after 3 (A and C) and 6 (B and D) days as shown by decreasing CFSE fluorescence of mock-transduced (A and B) or p53 TCR-expressing (C and D) PBLs cocultured with Saos 2, H2087, MDA-MB-231, or no target.

FIGURE 7

Functional reactivity of p53 TCR-transduced lymphocytes. Expression of CD107a (degranulation marker) was detected by FACS analysis on the surface of human PBLs expressing the p53 TCR after 2 h coculture with the indicated tumor cell line (control: A–D; p53⁺/HLA-A2⁺ : E–K). The percentage of p53 TCR⁺/CD107a⁺ cells was as shown.

FIGURE 8

Specific killing of tumor cell lines. Human PBLs expressing the p53 TCR were cocultured for 6 h with the indicated tumor cell lines previously labeled with ⁵¹Cr. Specific lysis was measured at the E:T ratio indicated using: [(specific release − spontaneous release)/(total release − spontaneous release)]. A, The HLA-A2⁺/p53⁺ cell lines used include: Saos 2/*143 (▴), MDA-MB-231 (×), H2087 (♦), BE-3 (•), in addition to the two controls MDA 386 (*, HLA-A2⁻/p53⁺) and Saos 2 (▪, HLA-A2⁺/p53). B, We compared the lysis activity of p53 TCR-transduced cells (filled symbols) with mock-transduced cells (open symbols) on the following targets: HLA-A2⁺/p53⁺ H2087 (diamond), Saos 2 (square), normal renal cells ELW91 (triangle), OKT3/IL-2 activated PBLs (circle), and normal resting (3 wk poststimulation) (ellipse) T cells.

FIGURE 9

Specific recognition of fresh human tumor cells. Human PBLs expressing the p53 TCR were cocultured for 16 h with fresh human tumor cells (melanoma) from different patients. The concentration of IFN-γ secreted in the medium was measured using an ELISA procedure.

FIGURE 10

Recognition and killing of tumor by transduced CD4⁺ cells. A, Different subsets (CD8^+, CD4^+, or bulk population) of human PBLs expressing the p53 TCR were cocultured for 16 h with the indicated peptide-pulsed APCs, tumor cell lines, or fresh human tumor samples (patient no. 7, HLA-A2⁺ and patient no. 12, HLA-A2⁻). The concentration of IFN-γ secreted in the medium was measured by ELISA. B–E, Different T cell subsets (CD8^+, CD4^+, or bulk population) of human PBLs expressing the p53 TCR were cocultured for 6 h with different tumor cell lines previously labeled with ⁵¹Cr. Specific lysis of Saos 2 (B), H2087 (C), MDA-MB-231 (D), JY (E) was measured at the E:T ratio indicated using: [(specific release − spontaneous release)/(total release − spontaneous release)].