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. 2008 May;57(5):693-700.
doi: 10.1007/s00262-007-0409-x. Epub 2007 Nov 15.

MYCN as a target for cancer immunotherapy

Nourredine Himoudi  1 , Mengyong Yan, Antigoni Papanastasiou, John Anderson
Affiliations

MYCN as a target for cancer immunotherapy (V体育官网入口)

Nourredine Himoudi et al. Cancer Immunol Immunother. 2008 May.

Abstract

MYCN is a potential target for cancer immunotherapy by virtue of its overexpression in numerous human malignancies and its functional role in tumour progression. Here we show limited expression of MYCN in normal human tissues indicating that anti-MYCN immune responses are unlikely to cross react with self tissues. An HLA-A2 restricted ten amino acid peptide epitope from MYCN, VILKKATEYV, was used to stimulate cytotoxic T cell lines from the peripheral blood of normal blood donors, and from a patient with MYCN amplified neuroblastoma. Strong and specific activity was seen against each MYCN overexpressing cell line and against autologous tumour cells VSports手机版. We generated two CTL clones capable of killing cells pulsed with as low as 0. 5 nM of VIL peptide. Therefore strong and specific immune responses against MYCN expressing tumours are possible in patients with the most common HLA class 1 type in the Caucasian population. .

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Figures

Fig. 1
Fig. 1
Differential MYCN expression in normal and cancer tissues. a MYCN expression at the RNA level measured by quantitative RT-PCR in a panel of normal human tissues and some rhabdomyosarcoma and neuroblastoma cell lines. b Immunoblot analysis of MYCN expression in cells lines used in cytotoxicity assays. IMR32 lysate was derived separately from nuclear and cytoplasmic extracts and each IMR32 sample reflects an equivalent number of cells. c HLA-A2 epression as determined by flow cytometry; pale line is isotype control, thick line is HLA-A2 staining. IMR32 cell line was treated with IFN-γ (5,000 U/ml) for 24 h before staining
Fig. 2
Fig. 2
T cell lines from two separate donors directed against the VIL epitope are capable of the specific recognition of MYCN expressing cells. a T2 binding assay in which duplicate VIL peptides were tested against positive (Flu marix) and negative (irrelevant mouse peptide) controls. b Anti-VIL T cell lines from two separate HLA-A2 positive donors are equally specifically reactive against VIL pulsed T2 cells in IFN-γ ELISPOT assay. Error bars reflect standard error of the mean of triplicate assays. c Transient transfection assay in which A2 positive 293T cells were cotransfected with MYCN and ornithine carboxylase promoter–luciferase reporter. MYCN activity in lysates is quantified in terms of relative light units following normalisation of the luciferase assay. Error bars are SEM of triplicates. d IFN-γ release following coculture of anti-VIL T cell lines with 293T target cells depicted in c or with MYCN positive SKNAS cells or MYCN negative RH18 cells
Fig. 3
Fig. 3
Anti-VIL T cell lines from normal donors can specifically kill MYCN expressing HLA-A2 positive target cells. a Standard chromium release assay at the E:T ratios indicated. b Specific killing of VIL pulsed T2 cells is inhibited by anti-MHC class 1 and anti-CD8 antibodies but not by equivalent concentration of isotype control antibody. Error bars indicate standard error of the mean of triplicate estimates of inhibition of a single T cell line
Fig. 4
Fig. 4
Anti-VIL is a component of an anti-tumour response in one patient with a MYCN expressing neuroblastoma. a CTL line derived from the peripheral blood of an HLA-A2 positive MYCN amplified neuroblastoma patient is capable of the specific lysis of autologous and MYCN positive tumour cell lines RH30 and IMR32. Error bars indicate standard error of the mean. b PBMC from this patient before and after DC/autologous lysate vaccination, were co-cultured with 20 μg/ml of autologous tumour lysate (TL) or irrelevant tumour lysate (irrTL, prepared from TC32, Ewing sarcoma HLA-A2+ tumour cell line) pulsed or not onto autologous DCs for 48 h. c 1 × 105 PBMC taken pre-vaccination, or post vaccination, were co-cultured with 10 μM VIL or an irrelevant peptide (KLTEARVQV peptide, a HLA-A2 restricted epitope derived from human PAX3 protein, Irr peptide), or co-cultured with 104 peptide pulsed DC. Data are means of triplicate estimations and error bars are SEM
Fig. 5
Fig. 5
Specific CTL clones were raised against MYCN derived peptide; clone VIL/C1 is shown at the top and VIL/C2 is shown at the bottom. a Pentamer staining of CTL clone VIL/C1 and VIL/C2. b Killing of T2 cell pulsed with 10 μM of VIL peptide by specific clones VIL/C1 top and VIL/C2 bottom. c VIL peptide titration, T2 target cells were pulsed with different concentration of VIL epitope and co-culture with effector clones VIL/C1 top and VIL/C2 bottom at effector to target ratio of 10:1. Data are means of triplicate estimations and error bars are SEM. d Killing of IMR32 cell line by clone VIL/C2. IMR32 cells were pre-cultured for 48 h with IFN-γ prior to the assay, E:T ratio shown is 50:1
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