Immunization of patients with the hTERT:540-548 peptide induces peptide-reactive T lymphocytes that do not recognize tumors endogenously expressing telomerase

Abstract

Purpose: Telomerase is an attractive target antigen for cancer immunotherapies because it is expressed in >85% of human tumors but is rarely found in normal tissues. A HLA-A*0201-restricted T-cell epitope was previously identified within telomerase reverse transcriptase hTERT:540-548. This peptide was reported to induce CTL that recognized tumor cells and transfectants that endogenously expressed telomerase. Therefore, we initiated a clinical protocol to evaluate the therapeutic and immunological efficacy of this peptide.

Experimental design: Fourteen patients with metastatic cancers were vaccinated with hTERT:540-548 emulsified in incomplete Freund's adjuvant.

Results: In 7 patients, peripheral blood mononuclear cells collected after immunization recognized hTERT:540-548, whereas those collected before vaccination did not. However, none of these CTLs recognized tumors that endogenously expressed telomerase, and none of the patients had an objective clinical response. Several highly avid T-cell clones were generated that recognized T2 cells pulsed with <or=1 nm hTERT:540-548, but none of these recognized HLA-A*0201(+) hTERT(+) tumors or cells transduced with the human telomerase reverse transcriptase (hTERT) gene. Also, an antibody specific for hTERT:540-548/HLA-A*0201 complexes stained peptide-pulsed cells but not telomerase(+) tumors.

Conclusions: Our results are discordant with previous studies and those of a clinical trial that claimed peripheral blood mononuclear cells from patients vaccinated with peptide-pulsed dendritic cells lysed hTERT(+) tumors. However, our findings are consistent with a previous study that demonstrated that the hTERT:540-548 peptide is cleaved in the proteasome. These results suggest that hTERT:540-548 is not presented on the surfaces of tumor cells in the context of HLA-A*0201 and will not be useful for the immunotherapy of patients with cancer.

Fig. 1

Telomerase enzyme activity in cell lysates as evaluated using a telomeric repeat amplification protocol assay followed by detection of the telomerase products by ELISA. The amount of cell lysate was normalized for the same number of cells in each assay. Also shown is the expression of HLA-A*0201 as evaluated by fluorescence-activated cell sorting and the presence of human telomerase reverse transcriptase (hTERT) mRNA as determined by reverse transcriptase-PCR. A, TSR8 PCR template, negative/contamination PCR control (buffer), positive control cells (+ control), and cell lines. B, WI-38 cells transduced with a recombinant adenovirus encoding hTERT at two different multiplicities of infection (MOI).

Fig. 2

HLA-A*0201/hTERT:540-548 tetramer analysis of peripheral blood mononuclear cells from patient 11 after 16 cycles of immunizations with hTERT:540-548 and after 12 days of in vitro culture with peptide. Cytokine secretion profiles from this T-cell population can be found in Table 2 (patient 11 after immunization), Table 3 (patient 11 post), and Table 4 (patient 11 bulk experiment 1). A, negative control phycoerythrin-conjugated HLA-A*0201/HTLV-1 tax peptide L11 tetramer versus FITC-labeled antihuman CD8. B, phycoerythrin-conjugated HLA-A*0201/hTERT:540-548 tetramer versus FITC-labeled antihuman CD8.

Fig. 3

Correlation of cytolysis and cytokine secretion by hTERT:540-548-reactive T-cell lines. T-Cell lines from 2 patients with metastatic melanoma were stimulated with hTERT:540-548 in the microculture format. After one restimulation (∼ day 14), the microcultures were evaluated for specific peptide recognition via cytokine secretion, and those that were positive were restimulated at least two additional times in 24-well plates. Seven to 14 days after the last restimulation, the T-cell populations were evaluated for recognition of peptide and hTERT⁺ cell lines in both cytolysis (A and B) and IFN-γ secretion assays (C and D).

Fig. 4

Staining of cell lines with Fab fragments (4G9) against HLA-A*0201/hTERT:540-548 complexes. A, T2 cells were pulsed with various concentrations of hTERT:540-548 and were subsequently stained with 4G9 Fabs. The dashed line indicates the mean fluorescence intensity (MFI) of background staining of 4G9 on T2 cells pulsed with an irrelevant control peptide [HBVc:18-27(23Y)]. B, T2 cells pulsed with 10 μg/ml hTERT:540-548 or HBVc:18-27(23Y) and B-cell lines were stained with 4G9 Fabs. The dashed line indicates background MFI of 4G9 staining of HLA-A*0201^- C1R cells. C, T2 cells pulsed with 100 μg/ml hTERT:540-548 or HBVc:18-27(23Y), melanoma cell lines, and 293 cells were stained with 4G9 Fabs. The dashed line indicates background MFI of 4G9 staining of T2 cells pulsed with HBVc:18-27(23Y).