Spontaneous T-cell responses against peptides derived from the Taxol resistance-associated gene-3 (TRAG-3) protein in cancer patients

Abstract

Expression of the cancer-testis antigen Taxol resistance-associated gene-3 (TRAG-3) protein is associated with acquired paclitaxel (Taxol) resistance, and is expressed in various cancer types; e.g., breast cancer, leukemia, and melanoma. Thus, TRAG-3 represents an attractive target for immunotherapy of cancer. To identify HLA-A*02.01-restricted epitopes from TRAG-3, we screened cancer patients for spontaneous cytotoxic T-cell responses against TRAG-3-derived peptides. The TRAG-3 protein sequence was screened for 9mer and 10mer peptides possessing HLA-A*02.01-binding motifs. Of 12 potential binders, 9 peptides were indeed capable of binding to the HLA-A*02.01 molecule, with binding affinities ranging from strong to weak binders. Subsequently, lymphocytes from cancer patients (9 breast cancer patients, 12 melanoma patients, and 13 patients with hematopoietic malignancies) were analyzed for spontaneous reactivity against the panel of peptides by ELISpot assay. Spontaneous immune responses were detected against 8 epitope candidates in 7 of 9 breast cancer patients, 7 of 12 melanoma patients, and 5 of 13 patients with hematopoietic malignancies. In several cases, TRAG-3-specific CTL responses were scattered over several epitopes. Hence, no immunodominance of any single peptide was observed. Furthermore, single-peptide responses were detected in 2 of 12 healthy HLA-A2(+) donors, but no responses were detectable in 9 HLA-A2(-) healthy donors or 4 HLA-A2(-) melanoma patients. The identified HLA-A*02.01-restricted TRAG-3-derived epitopes are targets for spontaneous immune responses in breast cancer, hematopoietic cancer, and melanoma patients. Hence, these epitopes represent potential target structures for future therapeutic vaccinations against cancer, possibly appropriate for strategies that combine vaccination and chemotherapy; i.e., paclitaxel treatment.

Fig. 1a–f

Overview of ELISpot results. PBLs from 12 healthy HLA-A*02.01-positive donors (a), 9 healthy HLA-A*02.01-negative donors (b), 4 HLA-A*02.01-negative melanoma patients (c), 9 HLA-A*02.01-positive breast cancer patients (d), 13 HLA-A*02.01-positive patients with hematopoietic malignancies (e), and 12 HLA-A*02.01-positive melanoma patients (f) were tested for spontaneous immune responses against the panel of TRAG-3–derived peptides capable of binding to HLA-A*02.01 molecules

Fig. 2

Spontaneous immune responses against TRAG-3–derived peptides in HLA-A2-positive breast cancer patients. Responses were detected by ELISpot, measuring IFN-γ secretion by T cells. Peptide-specific spontaneous responses were measured twice in triplicates. Columns represent the number of antigen-specific spots formed per 10⁵ T cells; bars indicate SD. Dark columns indicate spontaneous responses (defined as antigen-specific spots ± 1/2 SD >25 spots per 10⁵ cells)

Fig. 3

Spontaneous immune responses against TRAG-3–derived peptides in HLA-A2-positive hematopoietic patients. Responses were detected by ELISpot, measuring IFN-γ secretion by T cells. Due to limited patient material, it was not possible to analyze for reactivity against the full panel of peptides in all patients. Columns represent the number of antigen specific spots formed per 10⁵ T cells. Dark columns indicate spontaneous responses (defined as antigen specific spots ± 1/2 SD >25 spots per 10⁵ cells); bars indicate SD. CLL chronic lymphatic leukemia, MM multiple myeloma, LYM lymphoma

Fig. 4

Spontaneous immune responses against TRAG-3–derived peptides in HLA-A2-positive melanoma patients. Responses were detected by ELISpot, measuring IFN-γ secreted by T cells upon TCR stimulation. Due to limited patient material, it was not possible to analyze for reactivity against the full panel of peptides in all patients. Columns represent the number of antigen specific spots formed per 10⁵ T cells. Dark columns indicate spontaneous responses (defined as antigen specific spots ± 1/2 SD >25 spots per 10⁵ cells). Bars indicate SD. Lymphocytes of responding patients originated from infiltrated lymph nodes (LN), metastases (ME), or blood samples (PBL)