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Wt1-origin HLA-DR-binding antigen peptide

A technology for HLA-DRB1 and cancer antigens, applied in the field of HLA-DRB1*0405-binding antigen peptides

Active Publication Date: 2009-07-15
INT INST OF CANCER IMMUNOLOGY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports of WT1-derived peptides capable of binding different isoforms

Method used

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  • Wt1-origin HLA-DR-binding antigen peptide
  • Wt1-origin HLA-DR-binding antigen peptide
  • Wt1-origin HLA-DR-binding antigen peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] 1. Preparation of Dendritic Cells

[0193] Extraction of HLA-DRB1 from HLA-DRB1 by density gradient centrifugation using Ficoll-Paque ＊ 0405-positive peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy volunteers. Divide the resulting 8 x 10 6 PBMCs were suspended in 2ml X-VIVO 15 containing 1% AB serum TM culture medium (Camblex), inoculated in a 6-well culture plate, and cultured for 2 hours. After culturing, non-adherent cells were removed and adherent cells were washed with Hanks solution. In X-VIVO 15 containing 1% AB serum, 1000U / ml IL-4 and 1000U / ml GM-CSF TM Culture adherent cells in culture medium. On days 2 and 4 of culture, half of the medium was replaced with fresh medium. On day 6, TNF-α was added to a final concentration of 100 U / ml. Cells that survived on day 7 were used as experimental dendritic cells.

[0194] 2. Preparation of CD4 positive T cells (helper T cells)

[0195] Blood obtained from the same healthy vol...

Embodiment 2

[0204] Establishment of CD4-positive T cell lines specific for WT1 peptide

[0205] Dendritic cells prepared by a method similar to Example 1 were prepared in 10 4 Cells / well were seeded in 96-well plates, and then 10 3 Cells / well seeded as WT1 332-347 CD4 positive T cells induced by peptide (SEQ ID NO: 24). As for the medium, X-VIVO 15 containing 1% AB serum, 20 U / ml IL-2 and 5 μg / ml PHA was used TM Medium. The CD-4 positive T cell line was established by continuous culture and named "G2 cell line". The response of the G2 cell line to dendritic cells stimulated with the peptide was determined by a method similar to that of claim 1 . The results are shown in figure 2 middle. When used with WT1 332-347 The G2 cell line showed a proliferative response when co-cultured with peptide-stimulated dendritic cells but not when co-cultured with non-peptide-stimulated dendritic cells.

[0206] These results suggest that the G2 cell line is a WT1-specific 332-347 Peptide CD4 ...

Embodiment 3

[0208] WT1 peptide antigen presentation to HLA-DR molecules

[0209] In a manner similar to Example 1, HLA-DRB1 was extracted from HLA-DRB1 by density gradient centrifugation using Ficoll-Paque ＊ 0405-positive peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy volunteers. Then, with 10 7 Cells / well PBMCs were seeded in 24-well plates. As the medium, RPMI1640 medium containing 10% FCS and 55 µM 2ME was used. After addition of a medium containing Epstein-Barr virus (EBV), culture was continued for another 4 weeks to establish a B-cell line transformed with EBV, which was named "B-LCL(-) cells". EBV was prepared from the culture supernatant of B95-8 (JCRBCell Bank No. 9123), which is an EBV-producing cell line. Adjust B-LCL(-) cells to 3 x 10 7 cells / mL, and a medium containing a virus expressing the WT1 gene was added thereto, followed by addition of polypropylene (final concentration, 8 μg / mL), and the mixture was added to a 24-well plate ...

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Abstract

The present invention provides a WT1-derived HLA-DRB1*0405-binding antigen peptide, a polynucleotide encoding said peptide, a helper T cell inducer comprising said peptide or polynucleotide, and the like. It is related to a partial peptide consisting of 10 - 25 contiguous amino acids in the amino acid sequence of human WT1 shown in SEQ ID NO: 1, which binds to HLA-DRB1*0405 and induces helper T cells, a polynucleotide encoding said peptide, or a helper T cell inducer comprising said peptide or polynucleotide.

Description

technical field [0001] The present invention relates to WT1 derived HLA-DRB1 ＊ 0405 - Conjugated antigenic peptide. Background technique [0002] The WT1 gene (Wilms' tumor gene 1) has been identified as one of the causative genes of Wilms' tumor, ie, pediatric renal tumor (Cell 60:509, 1990, Nature 343:774, 1990). The WT1 gene encodes the transcription factor WT1, which plays an important role in many processes such as cell proliferation, differentiation, apoptosis and tissue development (Int. Rev. Cytol. 181: 151, 1998). Originally the WT1 gene was described as a tumor suppressor gene. However, follow-up studies revealed that the WT1 gene is highly expressed in leukemia and a variety of solid tumors including lung and breast cancers, indicating that the WT1 gene more precisely plays an oncogenic role in promoting cancer growth. In addition, it was demonstrated that when HLA-A was stimulated in vitro with a WT1-derived peptide ＊ 0201 or HLA-A ＊ 2402-positive peripheral...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09A61K35/12A61K35/76C12N5/00A61P35/00C07K7/04C07K14/705C07K14/82C07K16/30C07K16/32C07K19/00C12N1/15C12N1/19C12N1/21A61K38/04C12P21/02A61K38/00A61P37/04C07K7/08C07K14/47C12N5/12
CPCC07K14/4748A61K39/0011A61K38/00A61P35/00A61P37/04A61K39/001153C07K14/47C12N15/11
Inventor 杉山治夫
Owner INT INST OF CANCER IMMUNOLOGY INC
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