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EGFR T790M mutation as a possible target for immunotherapy; identification of HLA-A*0201-restricted T cell epitopes derived from the EGFR T790M mutation.

Yamada T, Azuma K, Muta E, Kim J, Sugawara S, Zhang GL, Matsueda S, Kasama-Kawaguchi Y, Yamashita Y, Yamashita T, Nishio K, Itoh K, Hoshino T, Sasada T - PLoS ONE (2013)

Bottom Line: Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non-small cell lung cancers (NSCLCs).Currently there are no effective treatment options for patients with this resistance mutation.When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Immunotherapy, Kurume University School of Medicine, Kurume, Fukuoka, Japan ; Department of Surgery, Fukuoka University School of Medicine, Fukuoka, Fukuoka, Japan.

ABSTRACT
Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non-small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively. Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them [1 of 7 (14%) vs 9 of 15 (60%); chi-squared test, p  =  0.0449], indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR-TKI resistance with the secondary EGFR T790M resistance mutation in NSCLC patients.

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Cross-reactivity of T790M-5-stimulated T cells against the wild-type or T790M-8 peptide.The T790M-5 stimulated PBMCs (2×104 cells/well) were examined for reactivity against T2 cells (1×104 cells/well) pulsed with T790M-5, WT-5, or T790M-8 at the indicated concentrations by IFN-γ ELISPOT assay. The assays were carried out in duplicate wells. Representative wells (A) and the means of spot numbers from duplicate wells (B) are shown. The experiments were repeated with blood from two different donors, and a representative result is shown.
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pone-0078389-g004: Cross-reactivity of T790M-5-stimulated T cells against the wild-type or T790M-8 peptide.The T790M-5 stimulated PBMCs (2×104 cells/well) were examined for reactivity against T2 cells (1×104 cells/well) pulsed with T790M-5, WT-5, or T790M-8 at the indicated concentrations by IFN-γ ELISPOT assay. The assays were carried out in duplicate wells. Representative wells (A) and the means of spot numbers from duplicate wells (B) are shown. The experiments were repeated with blood from two different donors, and a representative result is shown.

Mentions: Since T790M-5 was different from the corresponding wild-type peptide, WT-5, by only a single amino acid sequence, we examined whether T cell lines established after repeated stimulation with T790M-5 could show reactivity against the corresponding wild-type peptide WT-5 (Figure 4). When the T790M-5-stimulated T cell lines were stimulated by a higher dose (1 µg/ml) of peptides for ELISPOT assay, the wild-type peptide WT-5 induced apparent, but weaker antigen-specific T cell responses, compared to the mutant peptide T790M-5. When a lower dose (10 ng/ml) of peptides was used for the assay, the T790M-5-stimulated T cell lines responded only to T790M-5, but not to WT-5. This finding suggested that the T cells established by stimulation with the mutant peptide T790M-5 possessed a substantial, but weaker (around 100 times less) cross-reactivity to the corresponding wild-type peptide. Similarly, the T cell lines established by T790M-5 stimulation possessed an apparent, but weaker (around 100 times less) cross-reactivity to the T790M-8 peptide, which has one additional amino acid extension at the N-terminal of T790M-5 (Figure 4). We did not examine the cross-reactivity of T790M-7-stimulated T cell lines, since the corresponding wild-type peptide, WT-7, could not bind to HLA-A2 (Table 1).


EGFR T790M mutation as a possible target for immunotherapy; identification of HLA-A*0201-restricted T cell epitopes derived from the EGFR T790M mutation.

Yamada T, Azuma K, Muta E, Kim J, Sugawara S, Zhang GL, Matsueda S, Kasama-Kawaguchi Y, Yamashita Y, Yamashita T, Nishio K, Itoh K, Hoshino T, Sasada T - PLoS ONE (2013)

Cross-reactivity of T790M-5-stimulated T cells against the wild-type or T790M-8 peptide.The T790M-5 stimulated PBMCs (2×104 cells/well) were examined for reactivity against T2 cells (1×104 cells/well) pulsed with T790M-5, WT-5, or T790M-8 at the indicated concentrations by IFN-γ ELISPOT assay. The assays were carried out in duplicate wells. Representative wells (A) and the means of spot numbers from duplicate wells (B) are shown. The experiments were repeated with blood from two different donors, and a representative result is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818324&req=5

pone-0078389-g004: Cross-reactivity of T790M-5-stimulated T cells against the wild-type or T790M-8 peptide.The T790M-5 stimulated PBMCs (2×104 cells/well) were examined for reactivity against T2 cells (1×104 cells/well) pulsed with T790M-5, WT-5, or T790M-8 at the indicated concentrations by IFN-γ ELISPOT assay. The assays were carried out in duplicate wells. Representative wells (A) and the means of spot numbers from duplicate wells (B) are shown. The experiments were repeated with blood from two different donors, and a representative result is shown.
Mentions: Since T790M-5 was different from the corresponding wild-type peptide, WT-5, by only a single amino acid sequence, we examined whether T cell lines established after repeated stimulation with T790M-5 could show reactivity against the corresponding wild-type peptide WT-5 (Figure 4). When the T790M-5-stimulated T cell lines were stimulated by a higher dose (1 µg/ml) of peptides for ELISPOT assay, the wild-type peptide WT-5 induced apparent, but weaker antigen-specific T cell responses, compared to the mutant peptide T790M-5. When a lower dose (10 ng/ml) of peptides was used for the assay, the T790M-5-stimulated T cell lines responded only to T790M-5, but not to WT-5. This finding suggested that the T cells established by stimulation with the mutant peptide T790M-5 possessed a substantial, but weaker (around 100 times less) cross-reactivity to the corresponding wild-type peptide. Similarly, the T cell lines established by T790M-5 stimulation possessed an apparent, but weaker (around 100 times less) cross-reactivity to the T790M-8 peptide, which has one additional amino acid extension at the N-terminal of T790M-5 (Figure 4). We did not examine the cross-reactivity of T790M-7-stimulated T cell lines, since the corresponding wild-type peptide, WT-7, could not bind to HLA-A2 (Table 1).

Bottom Line: Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non-small cell lung cancers (NSCLCs).Currently there are no effective treatment options for patients with this resistance mutation.When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Immunotherapy, Kurume University School of Medicine, Kurume, Fukuoka, Japan ; Department of Surgery, Fukuoka University School of Medicine, Fukuoka, Fukuoka, Japan.

ABSTRACT
Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non-small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively. Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them [1 of 7 (14%) vs 9 of 15 (60%); chi-squared test, p  =  0.0449], indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR-TKI resistance with the secondary EGFR T790M resistance mutation in NSCLC patients.

Show MeSH
Related in: MedlinePlus