Identification of HLA-A2-restricted CTL epitopes of a novel tumour-associated antigen, KIF20A, overexpressed in pancreatic cancer - PubMed (original) (raw)

. 2011 Jan 18;104(2):300-7.

doi: 10.1038/sj.bjc.6606052. Epub 2010 Dec 21.

S Hirata, A Irie, S Senju, Y Ikuta, K Yokomine, M Harao, M Inoue, Y Tomita, T Tsunoda, H Nakagawa, Y Nakamura, H Baba, Y Nishimura

Affiliations

Identification of HLA-A2-restricted CTL epitopes of a novel tumour-associated antigen, KIF20A, overexpressed in pancreatic cancer

K Imai et al. Br J Cancer. 2011.

Abstract

Background: Identification of tumour-associated antigens (TAAs) that induce cytotoxic T lymphocytes (CTLs) specific to cancer cells is critical for the development of anticancer immunotherapy. In this study, we aimed at identifying a novel TAA of pancreatic cancer for immunotherapy.

Methods: On the basis of the genome-wide cDNA microarray analysis, we focused on KIF20A (also known as RAB6KIFL/MKlp2) as a candidate TAA in pancreatic cancer cells. The HLA-A2 (A*02:01)-restricted CTL epitopes of KIF20A were identified using HLA-A2 transgenic mice (Tgm) and the peptides were examined to check whether they could generate human CTLs exhibiting cytotoxic responses against KIF20A(+), HLA-A2(+) tumour cells in vitro.

Results: KIF20A was overexpressed in pancreatic cancer and in some other malignancies, but not in their non-cancerous counterparts and many normal adult tissues. We found that KIF20A-2 (p12-20, LLSDDDVVV), KIF20A-8 (p809-817, CIAEQYHTV), and KIF20A-28 (p284-293, AQPDTAPLPV) peptides could induce HLA-A2-restricted CTLs in HLA-A2 Tgm without causing autoimmunity. Peptide-reactive human CTLs were generated from peripheral blood mononuclear cells of HLA-A2(+) healthy donors by in vitro stimulation with the three peptides, and those CTLs successfully exhibited cytotoxic responses to cancer cells expressing both KIF20A and HLA-A2.

Conclusion: KIF20A is a novel promising candidate for anticancer immunotherapeutic target for pancreatic cancers.

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Figures

Figure 1

Figure 1

The relative expression ratio of KIF20A mRNA in pancreatic cancer tissues (Pancreatic Ca) and in various normal tissues based on a cDNA microarray analysis. The KIF20A gene was overexpressed in all six pancreatic cancer tissues investigated, but barely expressed in many normal tissues, except for in the testis and thymus. The ratio of relative expression levels of KIF20A mRNA in six pancreatic cancer tissues to that of disease-free counterparts was calculated. For normal tissues, the average expression level of KIF20A mRNA in all tissues was assigned to be 1.0, and the relative expression level of KIF20A mRNA in each tissue was calculated. For pancreatic cancer, the expression levels of KIF20A mRNA in adjacent normal pancreatic tissues were assigned to be 1.0, and the relative expression levels of KIF20A mRNA in pancreatic cancers were calculated using specimens obtained from six patients with pancreatic cancer.

Figure 2

Figure 2

Expression of KIF20A mRNA and protein in human normal tissues, cancer cell lines, and pancreatic cancer tissues. (A) RT–PCR analysis of KIF20A mRNA expression in various normal tissues. KIF20A mRNA was not detected except for faint expression observed in the testis. (B) The expression of KIF20A mRNA and protein in various cancer cell lines investigated by RT–PCR (left) and western blot analyses (right). KIF20A mRNA was detected in all pancreatic cancer cell lines examined. The major bands at ∼100 kDa, which corresponds to the calculated molecular weight for KIF20A, are shown. It must be noted that SKHep1 and SKHep1/Mock expressed only a trace amount of the KIF20A protein, whereas KIF20A cDNA-transfected SKHep1, SKHep1/KIF20A, relatively expressed a large amount of the protein. (C) RT–PCR analyses of the KIF20A expression in pancreatic cancer tissues (T), and their normal counterparts (N). The expression of the KIF20A mRNA was detected in five of eight pancreatic tumour tissues, but little expression was detected in their normal counterparts. It must be noted that KIF20A mRNA expression was also detected in the peritoneum (patient 9 (Pt9)) and metastatic foci of the skin (patient 10 (Pt10)).

Figure 3

Figure 3

Immunohistochemical analyses of the KIF20A protein in (A) pancreatic cancer and in (B) various normal tissues. (Panel A) A strong staining of KIF20A was mainly observed at the cytoplasm and nuclei of cancer cells in six of nine cases (representative patients 5, 6, and 9 are shown), whereas a very weak staining was observed in acinar cells and in the normal ductal epithelium of their normal adjacent pancreatic tissues. A pancreatic cancer tissue without KIF20A expression is also shown (Patient 3). A similar strong staining was observed in the metastatic foci of the peritoneum (Patient 9). (Panel B) KIF20A was not detected in the normal brain, lung, liver, kidney, stomach, small intestine, colon, spleen, skeletal muscle, skin, thymus, and testis. Little staining was detected in tumour-forming pancreatitis. Positive staining signals are seen as brown. The scale bars represent 100 _μ_m.

Figure 4

Figure 4

Identification of HLA-A2-restricted mouse CTL epitopes of human KIF20A using HLA-A2 Tgm. (A) HLA-A2 Tgm were immunised with syngeneic BM-DCs pulsed with the peptide mixtures as described in the ‘Materials and Methods’ section, and CD4− spleen cells were stimulated with BM-DCs pulsed with or without each peptide for 6 days. INF-_γ_-producing CTLs were detected by an ELISPOT assay. KIF20A-2 (LLSDDDVVV), KIF20A-8 (CIAEQYHTV), and KIF20A-28 (AQPDTAPLPV) peptides indicated by the circles were shown to induce peptide-reactive CTLs. These assays were performed twice with similar results. (B) Immunohistochemical staining with anti-CD4 or anti-CD8 mAb in tissue specimens of HLA-A2 Tgm immunised with the KIF20A-8 peptide. After two-times vaccinations, these specimens were removed and analysed.

Figure 5

Figure 5

Induction of KIF20A-specific human CTLs from PBMCs of three HLA-A2-positive healthy donors. (A) KIF20A peptide-reactive CTLs generated from PBMCs of three HLA-A2-positive healthy donors effectively killed T2 cells (HLA-A2+, TAP deficient) pulsed with each peptide but not those unpulsed or pulsed with irrelevant and HLA-A2-restricted CTL epitopes of HIV. (B) Human CTLs exhibited cytotoxicity to the KIF20A+, HLA-A2+ human pancreatic cancer cell line PANC1 and to the colon cancer cell line CaCo-2, but not to KIF20A+, HLA-A2− human pancreatic cancer cell line PK9. (C) The cytotoxicity of human CTLs was KIF20A specific. Those CTLs killed SKHep1/KIF20A, but not mock-transfected SKHep1 cells. Representative data are shown. (D) Human CTL responses were inhibited by anti-HLA-class I mAb (W6/32, IgG2a) but not by anti-HLA-DR mAb (H-DR-1, IgG2a). The target cells used were PANC-1cell (KIF20A+, HLA-A2+) and PK9 cell (KIF20A+, HLA-A2−). (Panels A–D) Representative data from one of the three donors with similar results are shown.

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References

    1. Cleary SP, Gryfe R, Guindi M, Greig P, Smith L, Mackenzie R, Strasberg S, Hanna S, Taylor B, Langer B, Gallinger S (2004) Prognostic factors in resected pancreatic adenocarcinoma: analysis of actual 5-year survivors. J Am Coll Surg 198: 722–731 - PubMed
    1. Echard A, Jollivet F, Martinez O, Lacapere JJ, Rousselet A, Janoueix-Lerosey I, Goud B (1998) Interaction of a Golgi-associated kinesin-like protein with Rab6. Science 279: 580–585 - PubMed
    1. Eloubeidi MA, Desmond RA, Wilcox CM, Wilson RJ, Manchikalapati P, Fouad MM, Eltoum I, Vickers SM (2006) Prognostic factors for survival in pancreatic cancer: a population-based study. Am J Surg 192: 322–329 - PubMed
    1. Firat H, Garcia-Pons F, Tourdot S, Pascolo S, Scardino A, Garcia Z, Michel ML, Jack RW, Jung G, Kosmatopoulos K, Mateo L, Suhrbier A, Lemonnier FA, Langlade-Demoyen P (1999) H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies. Eur J Immunol 29: 3112–3121 - PubMed
    1. Fukushima S, Hirata S, Motomura Y, Fukuma D, Matsunaga Y, Ikuta Y, Ikeda T, Kageshita T, Ihn H, Nishimura Y, Senju S (2009) Multiple antigen-targeted immunotherapy with alpha-galactosylceramide-loaded and genetically engineered dendritic cells derived from embryonic stem cells. J Immunother 32: 219–231 - PubMed

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