Cancer Letters

Cancer Letters

Volume 369, Issue 1, 1 December 2015, Pages 112-123
Cancer Letters

Original Articles
Enhanced anti-colon cancer immune responses with modified eEF2-derived peptides

https://doi.org/10.1016/j.canlet.2015.08.002Get rights and content

Highlights

  • The peptide from eEF2, P739–747 (RLMEPIYLV), elicits CTL responses in vivo and in vitro.

  • The modifications in P739–747 sequence improve the immunogenicity against eEF2.

  • Analog peptides with HLA-A*0201 anchor induces CTLs with cross-reactive to the native peptide.

  • The modified analogs inhibit tumor growth more efficiently than the native peptides.

Abstract

Eukaryotic elongation factor-2 (eEF2) is overexpressed in many human cancers and is an attractive target for cancer immunotherapy. The eEF2 derived polypeptides have been shown to be able to induce cytotoxic T lymphocytes from healthy donor. Here, we demonstrate the evidence indicating that modification of a segment of peptides from wild type eEF2-derived immunogenic peptides is able to further enhance its capacity of inducing antigen-specific cytotoxic T lymphocytes (CTLs) against colon cancer cells. Using peptide-MHC binding algorithms, potential HLA-A2.1-restricted epitopes capable of inducing specific CD8+ CTLs were identified. By analyzing HLA-A2.1 affinity and immunogenicity, we further identified one novel immunogenic peptide, P739–747 (RLMEPIYLV), that elicited specific CTL responses in HLA-A2.1/Kb transgenic mice and culture with peripheral blood lymphocytes from colon cancer patients. Furthermore, replacing certain amino acids (at positions 1, 3, 7) within the P739–747 sequence improved the immunogenicity against eEF2. Several analogs containing the auxiliary HLA-A*0201 anchor residues were able to stably bind to HLA-A*0201 and enhance CTL responses compared with the native sequence; two of them showed increased anti-tumor effects during the adoptive immunotherapy in vivo. Thus, these results support that modified immunogenic analogs are promising candidates for peptide-based cancer vaccination and immunotherapy.

Introduction

Cytotoxic T cell (CTL)-based specific immunotherapy is considered one of the most promising strategies for tumor therapy. The CD8+ CTLs are capable of lysing tumor cells by recognizing peptides derived from tumor-associated antigens (TAAs) presented on MHC class I molecules [1], [2]. Indeed, encouraging clinical responses have been observed in some cancer patients receiving peptide-based tumor immunotherapy [3], [4]. As most TAAs are self-antigens, antigen-specific CTL repertoires may be significantly reduced in the host during the negative selection process, leaving behind a T-cell repertoire that is poorly effective at mounting productive antitumor responses [5]. As a potential approach to enhance the immunogenicity of epitopes recognized by CTLs, modifications to the peptide sequences are applied to improve binding to MHC class I molecules [6], [7], [8], [9]. However, replacing residues that are directed toward the TCR (T cell receptor) can also improve epitope immunogenicity [10], [11], [12]. Some of these peptides with enhanced immunogenicity have been successfully used to immunize cancer patients, improve detection of antitumor immune responses, and reverse non-responsiveness to tumor antigens [10].

Eukaryotic elongation factor-2 (eEF2) is previously identified as a novel tumor-associated antigen [13], [14]. Clinical specimens of cancer tissues show that eEF2 protein is highly expressed in human breast, prostate, lung, gastric and colorectal carcinoma tissues, but not in normal tissues, as examined by immunohistochemical analysis [13], [14], indicating eEF2 being an effective TAA target for immunotherapy. Oji et al. [14] have shown that eEF2-derived 9-mer peptides, EF786 (eEF2 786–794 aa) and EF292 (eEF2 292–300 aa), elicited cytotoxic T lymphocyte (CTL) responses in peripheral blood mononuclear cells (PBMCs) from an HLA-A*24:02- and an HLA-A*02:01-positive healthy donor, respectively, in an HLA-A-restricted manner. Following this line of study, we investigated whether additional eEF2-derived peptides could elicit CTL response, and whether the CTL response by such eEF2-derived peptides could be further enhanced by peptide modifications. Here, we identified a nonamer peptide derived from the eEF2 protein, designated as P739–747 (RLMEPIYLV), which possessed the ability to provoke a peptide-specific, HLA-A2.1-restricted CTL response in HLA-A2.1/Kb transgenic (Tg) mice in vivo as well as in peripheral blood lymphocytes (PBLs) from HLA-matched colon cancer patients using dendritic cells (DCs) pre-pulsed with the peptides in vitro. In addition, amino acid substitution at HLA-A*0201-binding anchor positions in the native peptide enhanced the HLA-A*0201-binding affinity and immunogenicity of these modified peptides. These results provide the first evidence indicating the potential clinical application of this eEF2-derived analog in peptide-mediated immunotherapy for eEF2-expressing tumors.

Section snippets

Synthetic peptides

All peptides utilized in this study were synthesized by GL Biochem (Shanghai, China) using fluorenylmethoxycarbonyl chemistry and purified to more than 95% by reversed phase high-performance liquid chromatography (HPLC), as confirmed by mass spectrometry. The lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO), diluted with phosphate-buffered saline (PBS; pH 7.4) at a concentration of 10 mM, and stored in aliquots at −80 °C. The amino-acid sequences and the predicted score for

Selection of potential eEF2-derived peptides binding to HLA-A*0201 molecules

Many types of human tumors overexpress the eEF2 protein compared to the related normal tissues, making it a potential TAA for immunotherapy. To begin more fully assessing this potential in terms of eliciting an immunogenic response in the host, we attempted to screen the eEF2 amino acid sequence for the presence of HLA-A*0201-binding motifs using computer-assisted analysis. We selected and synthesized 6 candidate nonameric peptides with the highest estimated half-life of dissociation from

Discussion

In this study, we show that immunogenic peptide, P739–747 (RLMEPIYLV), can elicit specific CTL responses in both HLA-A2.1/Kb transgenic mice and cell culture with peripheral blood lymphocytes from colon cancer patients. Furthermore, peptide modification (at positions 1, 3, 7) within the P739–747 sequence further enhanced the immunogenicity against eEF2. Analogs containing the auxiliary HLA-A*0201 anchor residues can bind to HLA-A*0201 and enhance CTL responses compared with the native sequence;

Conclusion

This study characterized P739–747 as a novel HLA-A*0201-restricted, immunogenic CD8+ T-cell epitope derived from eEF2 naturally processed and presented by colon cancer cells. We found that replacing secondary anchor residues in P739–747 yielded new sequences with enhanced in vivo and in vitro immunogenicity, as shown in the HLA-A2.1/Kb Tg mouse models and in human cells. These results collectively provide the first evidence indicating that the modified eEF2 peptides are potentially able to

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We acknowledge Dr. Changyou Li and Dr. Peng Zhao for helpful discussions. We also thank Danni Zhu and Qingming Guo for their assistance in experimental procedures. This work is partially supported by NIH RO1 grant CA152313 to JJL. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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