Diagnosis method and therapeutic method for cancer

A diagnostic agent and cancer vaccine technology, applied in gene therapy, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of high expression and no detection of HERV-Henv gene

Inactive Publication Date: 2011-10-12
KEIO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Among the HERV-H gene clusters, it has been reported that the HERV-H env gene is highly expressed in the placenta, skeletal muscle, spleen, and thymus, and no expression was detected in other normal tissues

Method used

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  • Diagnosis method and therapeutic method for cancer
  • Diagnosis method and therapeutic method for cancer
  • Diagnosis method and therapeutic method for cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060][Example 1] Expression of HERV-H env gene in people's normal tissues and tumor cells

[0061] This example shows that the endogenous retrovirus HERV-H env gene is basically not expressed in human normal tissues, but highly expressed in tumor cell lines and tumor tissues.

[0062] ==Gene Expression Analysis by RT-PCR==

[0063] Using RNeasykit (Qiagen Company), human normal tissues, various human tumor cell lines, and human colorectal carcinoma (refer to figure 1 ) was extracted from RNA and reverse transcribed with AMV to obtain cDNA. Then, using the following primers, the gene was amplified with iCycler (Biorad), and gene expression was detected by electrophoresis.

[0064] figure 1 The upper part of the graph shows the expression of the HERV-H env gene, and the lower part shows the expression of the GAPDH gene as an expression level control.

[0065] Primers for HERV-H env:

[0066] Forward 5'-GGATCCTCTACCTACATGTGTC-3' (SEQ ID NO: 6)

[0067] Reverse 5'-TCAAGGGAA...

Embodiment 2

[0073] [Example 2] Function of HERV-H env expressed in tumor cells and its inhibition

[0074] This example shows that HERV-H env is related to the invasion of cancer cells, and the inhibition of the expression of HERV-H env gene can inhibit the invasion of cancer cells.

[0075] (1) Forced expression of snail gene in Panc-1 cells

[0076] First, snail cDNA (CDS 71-865, 795 bp) was amplified by PCR from Panc-1 cells stimulated with TGF-beta, which is known as one of EMT inducers, and inserted into pcDNA3.1(+ ) in the restriction enzyme EcoR I-Xho I site of the plasmid vector (Invitrogen Company). It was introduced into the tumor cell line by electroporation, and after 2 weeks of culture, the drug-resistant cells were selected with G418 (2 mg / mL), and the cells were cloned.

[0077] figure 2 In the table, the phenotypes of the snail gene-introduced cell lines are summarized in the table.

[0078] Intrinsic expression of Snail protein was observed in Panc-1 cells as the par...

Embodiment 3

[0106] [Example 3] Induction of HERV-H env-specific CTL in mice altered using HLA-A24 expression gene

[0107] This example shows that the genetically modified mice (A24-Tg) expressing HLA-A24 (purchased from SLC, reference: International J. Cancer 100: 5565-5570) were immunized with HLA-A24-restricted HERV-H env peptide , 2002), HERV-H env has immunogenicity, that is, it can induce HERV-H env-specific CTL.

[0108]First, dendritic cell precursor cells were isolated from bone marrow cells of A24-Tg mice using LineagePanel Streptavidin Plus Magnetic Particles-DM (BD Biosciences), and cultured in the presence of GM-CSF (10ng / mL, Peprotech) for 6 God, divide it. In the presence of the peptide (10 μg / mL, synthesized by Invitrogen Co.) with the following sequence, the dendritic cells were cultured for 6 hours, and then subcutaneously in HLA-A24 expression gene-modified mice at 5×10 6 Inoculated mice were immunized. Furthermore, SAGE, which has been identified as a cancer testis ...

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Abstract

Disclosed are a diagnosis method and a therapeutic method for cancer, both of which utilize HERV-H env gene and protein. Particularly, cancer can be diagnosed by detecting the expression of HERV-H env gene, and a reagent used for the detection of the expression can be used as a diagnosing agent. Cancer can be treated by inhibiting the function of HERV-H env gene, and a reagent used for the inhibition of the function can be used as an anti-cancer agent. Alternatively, cancer can be treated by administering a peptide having a specific sequence contained in HERV-H env protein or the like, and the peptide or the like can be used as a cancer vaccine.

Description

technical field [0001] The present invention relates to a method for diagnosing cancer and a diagnostic agent used in the method, a method for treating cancer and an anticancer agent and a cancer vaccine used in the method. Background technique [0002] Human endogenous retrovirus (HERV) or other viral LTR-like sequences make up approximately 8% of genome DNA. In the human genome, more than 20 kinds of HERV have been identified, and the gag, pol, and env genes are basically mutated, but there are also mutations in the placenta, teratoma cell lines, germ cell-derived tumors, breast cancer cell lines, etc. Expressed genes detected. [0003] Among them, the HERV-H gene cluster forms the largest group, with about 100 copies of the full-length sequence, 800-900 copies of the sequence with deletion variation, and about 1000 copies of the LTR sequence. [0004] According to reports, in the HERV-H gene cluster, the expression of HERV-H env gene is high in placenta, skeletal muscle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K31/713A61K39/00A61K48/00A61P35/00C07K14/15C12N5/10C12N15/09C12Q1/68A61K35/12A61K35/14A61K35/15A61K35/17A61K35/76A61P35/02A61P37/04
CPCA61K31/713A61K39/00A61K39/12A61K39/21A61K2039/515A61K2039/5154A61K2039/53A61K47/6901A61P35/00A61P35/02A61P37/04C07K14/005C12N15/113C12N15/1132C12N2310/14C12N2740/10022C12N2740/10034C12Q1/6886C12Q2600/158
Inventor 工藤千惠河上裕
Owner KEIO UNIV
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