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Identification of a Promiscuous Epitope Peptide Derived From HSP70

Matsui, Hiroto, Hazama, Shoichi, Tamada, Koji, Udaka, Keiko, Irie, Atsushi, Nishimura, Yasuharu, Miyakawa, Tomoya, Doi, Shun, Nakajima, Masao, Kanekiyo, Shinsuke, Tokumitsu, Yukio, Shindo, Yoshitaro, Tomochika, Shinobu, Yoshida, Shin, Iida, Michihisa, Suzuki, Nobuaki, Takeda, Shigeru, Yamamoto, Shigeru, Yoshino, Shigefumi, Ueno, Tomio, Nagano, Hiroaki·2019·Journal of Immunotherapy
DOI 10.1097/cji.0000000000000274PMC PMC6727952
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Abstract

We previously found that heat-shock protein 70 (HSP70) is expressed on hepatocellular carcinoma cells and developed an HSP70 mRNA-transfected dendritic cell therapy for treating unresectable or recurrent hepatocellular carcinoma. The phase I trial was completed successfully. The purpose of this study is to identify a promiscuous epitope peptide derived from HSP70 for the purpose of developing a novel cancer peptide vaccine. Using a computational algorithm to analyze the specificity of previously reported major histocompatibility complex class I–binding peptides, we selected candidates that bound to >2 of the 3 HLA types. Twenty-nine HSP70-derived peptides (9-mers) that bound to HLA-class I was selected. The peptides were prioritized based on the results of peptide binding experiments. Using dendritic cells stimulated with the candidate peptide described previously as stimulators and CD8+ T cells as effectors, an ELISPOT assay was performed. Cytotoxicity of CD8 lymphocytes stimulated with the candidate peptides toward HSP70-expressing cancer cells was analyzed using an xCELLigence System. Peptides were administered to HLA-A 24 transgenic mice as vaccines, and peptide-specific T-cell induction was measured in vivo. We identified a multi-HLA-class I–binding epitope peptide that bound to HLA-A*02:01, *02:06, and *24:02 in vitro using an interferon-γ ELISPOT immune response induction assay. Cytotoxicity was confirmed in vitro, and safety and immune response induction were confirmed in vivo using HLA-A 24 transgenic mice. Our study demonstrated that the promiscuous HSP70-derived peptide is applicable to cancer immunotherapy in patients with HLA-A*24:02-positive, *02:01-positive, and *02:06-positive HSP70-expressing cancers.

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FIGURE 1 IFN-γ production by CTLs induced by heat-shock protein 70-derived peptides. A, IFN-γ production by CTLs from HCC patients induced by self-DCs pulsed with heat-shock protein 70-derived peptides. IFN-γ production was examined using an IFN-γ ELISPOT assay. Depending on the status of the allele, the HLA-A*24:02-restricted EBV peptide (TYGPVFMCL), HLA-A*02:01-restricted Flu A peptide (GILGFVFTL), and HLA-A*02:06-restricted EBV peptide (LTAGFLIFL) were used as positive controls. The HLA-A*24:02-restricted HIV (RYLRDQQLL), HLA-A*02:01-restricted HIV (SLYNTVATL), and HLA-A*02:06-restricted HIV (ATLEEMMTA) peptides were used as negative controls in the same manner. “Closed bars” indicate mean IFN-γ production by CTLs from HLA-A*24:02-positive HCC patients responding to self-DCs pulsed with indicated peptide, and “open bars” indicate mean IFN-γ production by CTLs from HLA-A*24:02-negative and HLA-A*02:01-positive or *02:06-positive HCC patients responding to self-DCs pulsed with indicated peptide. All experiments were performed in triplicate at a density of 5×104 cells/well. This experiment was performed at least 5 times in total for each peptide. Because the amount of sample was limited, experiments were not repeated beyond 5 times for peptides deemed unlikely candidates. However, to improve the accuracy for potential candidate peptides, additional experiments were repeated up to 26 times. Data represent the mean±SD of the IFN-γ release. *P<0.05. B, The representative data are shown. Peptide Y50-specific spots were increased in patients with HLA-A*24:02, *02:01, or *02:06. CTL indicates cytotoxic T lymphocyte; DC, dendritic cell; HCC, hepatocellular carcinoma; HIV, human immunodeficiency virus; IFN, interferon.
FIGURE 1 IFN-γ production by CTLs induced by heat-shock protein 70-derived peptides. A, IFN-γ production by CTLs from HCC patients induced by self-DCs pulsed with heat-shock protein 70-derived peptides. IFN-γ production was examined using an IFN-γ ELISPOT assay. Depending on the status of the allele, the HLA-A*24:02-restricted EBV peptide (TYGPVFMCL), HLA-A*02:01-restricted Flu A peptide (GILGFVFTL), and HLA-A*02:06-restricted EBV peptide (LTAGFLIFL) were used as positive controls. The HLA-A*24:02-restricted HIV (RYLRDQQLL), HLA-A*02:01-restricted HIV (SLYNTVATL), and HLA-A*02:06-restricted HIV (ATLEEMMTA) peptides were used as negative controls in the same manner. “Closed bars” indicate mean IFN-γ production by CTLs from HLA-A*24:02-positive HCC patients responding to self-DCs pulsed with indicated peptide, and “open bars” indicate mean IFN-γ production by CTLs from HLA-A*24:02-negative and HLA-A*02:01-positive or *02:06-positive HCC patients responding to self-DCs pulsed with indicated peptide. All experiments were performed in triplicate at a density of 5×104 cells/well. This experiment was performed at least 5 times in total for each peptide. Because the amount of sample was limited, experiments were not repeated beyond 5 times for peptides deemed unlikely candidates. However, to improve the accuracy for potential candidate peptides, additional experiments were repeated up to 26 times. Data represent the mean±SD of the IFN-γ release. *P<0.05. B, The representative data are shown. Peptide Y50-specific spots were increased in patients with HLA-A*24:02, *02:01, or *02:06. CTL indicates cytotoxic T lymphocyte; DC, dendritic cell; HCC, hepatocellular carcinoma; HIV, human immunodeficiency virus; IFN, interferon.CC BY-NC-ND · PubMed Central
FIGURE 2 Impedance traces of HepG2 tumor cells incubated alone or with varying concentrations of HLA-A*24:02-positive (A) or *02:01-positive (B) tumor-specific T cells. Each trace was calculated from triplicate data points collected every 5 minutes through the duration of the culture. C and D, Data for percent lysis of HepG2 cells in response to varying concentrations of heat-shock protein 70-derived peptide-specific T cells measured 5 hours after mixing of tumor and T cells, as determined using an impedance-based assay. C and D, indicate calculated data of (A) and (B), respectively. Percent lysis was determined using the following formula: (CI tumor only−[CI tumor+T cells])/(CI tumor only)×100. CI indicates cell index; E/T, effector to target.
FIGURE 2 Impedance traces of HepG2 tumor cells incubated alone or with varying concentrations of HLA-A*24:02-positive (A) or *02:01-positive (B) tumor-specific T cells. Each trace was calculated from triplicate data points collected every 5 minutes through the duration of the culture. C and D, Data for percent lysis of HepG2 cells in response to varying concentrations of heat-shock protein 70-derived peptide-specific T cells measured 5 hours after mixing of tumor and T cells, as determined using an impedance-based assay. C and D, indicate calculated data of (A) and (B), respectively. Percent lysis was determined using the following formula: (CI tumor only−[CI tumor+T cells])/(CI tumor only)×100. CI indicates cell index; E/T, effector to target.CC BY-NC-ND · PubMed Central
FIGURE 3 A, HLA-A24 Tg mice were immunized with phosphate buffered saline, adjuvant of 50 μg poly(I:C) plus 1 μg LAG-3-Ig, or 50 μg of heat-shock protein 70-derived peptide with adjuvant of 50 μg poly(I:C) plus 1 μg LAG-3-Ig once a week for 8 weeks. At 7 days after the last immunization, lymph nodes were harvested. Lymphocytes were cultured for 5 days with heat-shock protein 70-derived peptide (10 μg/mL) and then subjected to interferon-γ ELISPOT assay. Because of the small number of cells, lymphocytes from each group of mice were mixed and used in the ELISPOT assay. Experiments were carried out in triplicate. Data are reported as mean±SEM for each group. Lymphocytes of 3 mice immunized with peptide Y50 exhibited significantly greater interferon-γ production than those of the other groups. B, Representative data are shown. PBS indicates phosphate buffered saline.
FIGURE 3 A, HLA-A24 Tg mice were immunized with phosphate buffered saline, adjuvant of 50 μg poly(I:C) plus 1 μg LAG-3-Ig, or 50 μg of heat-shock protein 70-derived peptide with adjuvant of 50 μg poly(I:C) plus 1 μg LAG-3-Ig once a week for 8 weeks. At 7 days after the last immunization, lymph nodes were harvested. Lymphocytes were cultured for 5 days with heat-shock protein 70-derived peptide (10 μg/mL) and then subjected to interferon-γ ELISPOT assay. Because of the small number of cells, lymphocytes from each group of mice were mixed and used in the ELISPOT assay. Experiments were carried out in triplicate. Data are reported as mean±SEM for each group. Lymphocytes of 3 mice immunized with peptide Y50 exhibited significantly greater interferon-γ production than those of the other groups. B, Representative data are shown. PBS indicates phosphate buffered saline.CC BY-NC-ND · PubMed Central

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Research works printed but not linked: 24

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Metadata

Authors
Matsui, H., Hazama, S., Tamada, K., Udaka, K., Irie, A., Nishimura, Y., Miyakawa, T., Doi, S., Nakajima, M., Kanekiyo, S., Tokumitsu, Y., Shindo, Y., Tomochika, S., Yoshida, S., Iida, M., Suzuki, N., Takeda, S., Yamamoto, S., Yoshino, S., Ueno, T., & Nagano, H.
Venue
Journal of Immunotherapy
Publisher
Ovid Technologies (Wolters Kluwer Health)
Published
2019-09
Volume
42
Issue
7
Pages
244-250
ISSN
1524-9557
DOI
10.1097/cji.0000000000000274
PMC
PMC6727952
Cited by
11
References
1
Language
en
Licence
creativecommons.org/licenses/by-nc-nd/4.0/%20http://creativecommons.org/licenses/by-nc-nd/4.0/ (unspecified)

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CoChat. (2026). Citation check: Identification of a Promiscuous Epitope Peptide Derived From HSP70. https://app.cochat.ai/papers/10.1097/cji.0000000000000274
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Matsui, H., Hazama, S., Tamada, K., Udaka, K., Irie, A., Nishimura, Y., Miyakawa, T., Doi, S., Nakajima, M., Kanekiyo, S., Tokumitsu, Y., Shindo, Y., Tomochika, S., Yoshida, S., Iida, M., Suzuki, N., Takeda, S., Yamamoto, S., Yoshino, S., . . . Nagano, H. (2019). Identification of a Promiscuous Epitope Peptide Derived From HSP70. Journal of Immunotherapy. https://doi.org/10.1097/cji.0000000000000274