Means and methods for diagnosing cancer using an antibody which specifically binds to BRAF V600E

一种特异性、抗体的技术，应用在化学仪器和方法、抗受体/细胞表面抗原/细胞表面决定因子免疫球蛋白、特定肽等方向，能够解决肿瘤细胞百分数高、费时等问题

Active Publication Date: 2013-11-20
DEUTES KREBSFORSCHUNGSZENT STIFTUNG DES OFFENTLICHEN RECHTS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has certain limitations: the percentage of tumor cells in the tissue used for DNA extraction needs to be quite high
Too many impurities containing non-tumor cells will lead to false negative sequencing results
The DNA analysis procedure is time-consuming because first, the pathologist has to initially select suitable material for DNA extraction, which needs to be performed before DNA sequencing can be performed

Method used

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  • Means and methods for diagnosing cancer using an antibody which specifically binds to BRAF V600E
  • Means and methods for diagnosing cancer using an antibody which specifically binds to BRAF V600E
  • Means and methods for diagnosing cancer using an antibody which specifically binds to BRAF V600E

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Day 5: 20 μg of the immunogenic peptide dissolved in PBS was injected into mice at a ratio of 1:1 together with incomplete Freund's adjuvant.

[0067] Day 11: Inject 20 μg of immunogenic peptide dissolved in PBS into mice.

[0068] Day 18: 20 μg of immunogenic peptide dissolved in PBS was injected into mice.

[0069] On day 19, blood was drawn from the mice, and serum was isolated for Western blot testing of tumor proteins containing BRAF V600E or BRAF wild-type protein. On day 46, 20 μg of immunogenic peptide dissolved in PBS was injected into mice. On day 47, blood was drawn from the mice and serum was isolated for Western blot testing of tumor proteins containing BRAF V600E or BRAF wild-type protein. On day 48, the mice were sacrificed. Cubic lymph nodes were removed, cells were isolated and fused with SP2 / 0 lineage cells to generate hybridoma cells. Finally, the hybridoma cells are cultured, and single cells are picked and isolated to generate pure clones. Clon...

Embodiment 2

[0074] Clone 263 was deposited with "Deutsches Krebsforschungszentrum", Heidelberg, GERMANY under the Budapest Treaty on September 8, 2010 under the accession number DSM ACC 3091 in "DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH", 38124 Braunschweig, GERMANY.

[0075] Example 3: Antibody of clone 263 specifically targets and identifies hairy cell leukemia expressing BRAF V600E mutant polypeptide

[0076] All source materials were obtained from the Tissue Bank of the National Center for Tumor Diseases (NCT) and the Department of General Pathology, Institute for Pathology, Heidelberg. Tissue microarrays (TMA) constructed from paraffin-embedded tissues containing a total of 208 mature B-cell tumors, including plasmacytoma (n=26), follicular lymphoma (n= 34), diffuse large B-cell lymphoma (n=41), primary mediastinal B-cell lymphoma (n=25), mantle cell lymphoma (n=17), extranodal marginal zone lymphoma ( n=16), hairy cell leukemia (n=3) and Hodgkin's lymphoma ...

Embodiment 3

[0078] BRAF V600E can also be detected by direct DNA sequencing. DNA was extracted from formalin-fixed paraffin-embedded tissues of patients (n = 39) with HCL and SMZL. The primer sequences and direct sequencing procedure have been described previously (Schindler 2011, Acta Neuropathol 121: 397-405).

[0079] Screening of 208 human mature B-cell lymphomas for BRAF-V600E mutant activation with BRAF V600E-specific antibodies identified 2 positive samples in 3 cases of HCL. All other lymphoma cases were negative. Analysis of an additional 34 HCL bone marrow biopsies and 1 spleen biopsy not included in the initial tissue microarray analysis confirmed strong cytoplasmic expression of the mutant BRAF V600E protein in 31 of 35 (87%). Direct sequencing of genomic DNA confirmed the presence of the BRAF-V600E mutation in 24 (77%) of 31 immunohistochemically positive HCL cases. In seven bone marrow biopsies expressing BRAF-V600E, BRAF mutations could not be detected by direct sequenci...

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Abstract

The present invention relates to the field of diagnostic tests and diagnostic tools. Specifically, contemplated is to a method for diagnosing cancer in a sample of a subject suspected to suffer from cancer comprising contacting the sample with an antibody which specifically binds to the epitope characterized by SEQ ID NO 1 on a BRAF polypeptide under conditions which allow for binding of said antibody to the epitope and determining binding of the antibody to the epitope, whereby cancer is diagnosed. Further contemplated are antibodies which specifically bind to the epitope characterized by SEQ ID NO 1 on a BRAF polypeptide and a device or kit comprising such antibodies.

Description

[0001] The present invention relates to the field of diagnostic tests and diagnostic tools. In particular, contemplated is a method for diagnosing cancer in a sample of a subject suspected of suffering from cancer, the method comprising allowing said sample to specifically bind to an epitope on a BRAF polypeptide characterized by SEQ ID NO 1 The antibody is brought into contact with the epitope under conditions that allow the antibody to bind to the epitope, and the binding of the antibody to the epitope is determined, thereby diagnosing cancer. Also contemplated are antibodies that specifically bind to the epitope on the BRAF polypeptide characterized by SEQ ID NO 1 and devices or kits comprising such antibodies. background [0002] Cancer is the second leading cause of death in the United States after cardiovascular disease. One in three Americans develops cancer during his or her lifetime, and one in four Americans dies from cancer. Despite profound experience with tradit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/574C07K16/00
CPCC07K16/30G01N33/5088G01N33/6893G01N33/5743G01N33/543G01N33/57426C07K16/32
Inventor H.岑特格拉夫A.冯戴姆林D.卡佩尔
Owner DEUTES KREBSFORSCHUNGSZENT STIFTUNG DES OFFENTLICHEN RECHTS
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