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Antibodies Against Claudin 18.2 Useful in Cancer Diagnosis

An antibody and antigen technology, applied in the field of antibodies against claudin 18.2 for cancer diagnosis, which can solve problems such as expression limitations

Active Publication Date: 2018-05-18
ASTELLAS PHARMA INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, expression of CLDN18.2 in the normal stomach is restricted to differentiated short-lived gastric epithelial cells

Method used

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  • Antibodies Against Claudin 18.2 Useful in Cancer Diagnosis
  • Antibodies Against Claudin 18.2 Useful in Cancer Diagnosis
  • Antibodies Against Claudin 18.2 Useful in Cancer Diagnosis

Examples

Experimental program
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Effect test

preparation example Construction

[0158] Sample preparation is critical to maintain cell morphology, tissue architecture, and antigenicity of target epitopes. This requires proper tissue collection, fixation and sectioning. Usually paraformaldehyde is used for fixation. Depending on the purpose and thickness of the experimental sample, thin (approximately 4 to 40 μm) sections are cut from the tissue of interest, or it can be used whole if the tissue is not very thick and penetrable. Sectioning is typically accomplished by using a microtome and mounting the sections on slides.

[0159] Samples may require additional steps to render epitopes available for antibody binding, including deparaffinization and antigen retrieval. Typically, detergents (such as Triton X-100) are used in immunohistochemistry to reduce surface tension so that less reagent can be used to achieve better and more uniform coverage of the sample.

[0160] The direct method of immunohistochemical staining uses a labeled antibody that binds d...

Embodiment 1

[0255] Example 1: Production of Monoclonal Antibodies

[0256] The aim of this project is to generate mouse CLDN-18-specific monoclonal antibodies capable of detecting CLDN18.2-expressing tumor cells in gastric CA, esophageal CA, pancreatic CA, and lung CA FFPE tissues.

[0257] In order to generate highly specific, high affinity diagnostic CLDN18.2 antibodies, it is necessary to start an immunization regimen with a wide variety of different immunogens and adjuvants. During the project, approximately 100 mice (C57B1 / 6 and Balb / c) were vaccinated using multiple immunization strategies to elicit an α-CLDN18 immune response.

[0258] To prime the mouse immune system and overcome immune tolerance, we employed virus-like particles (VLPs), peptide conjugates, and recombinant proteins encoding different parts of human CLDN18.2 expressed as different expression partners ( tag) recombinant fusion protein.

[0259] In 13 different immunization strategies, the HIS-tagged C-terminal rec...

Embodiment 2

[0264] Example 2: Western Blot Screening of Monoclonal Hybridoma Supernatants

[0265] To answer the question of whether ELISA positive antibodies in the supernatants were able to bind to recombinant claudin 18 or protein lysates from stably transfected claudin 18 expressing HEK293 cells, Western blot analysis was performed. Amplification of antibodies capable of binding specifically to claudin 18 in Western blot analysis. Cells were frozen and antibodies were purified by MABselect (FPLC). Antibodies selected by Western blot screening were purified and evaluated by immunohistochemistry for their ability to bind their antigens in formalin-fixed paraffin-embedded tissue (FFPE).

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Abstract

The invention relates to antibodies against claudin 18.2 useful in cancer diagnosis. The invention relates to antibodies directed against an epitope located within the C-terminal portion of CLDN18.2 which are useful, for example, in diagnosing cancer and / or in determining whether cancer cells express CLDN18.2.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201380024247.0. The original application is the PCT international application PCT / EP2013 / 001331 filed on May 6, 2013, which entered the Chinese national phase on November 7, 2014. technical field [0002] The present invention relates to antibodies against claudin 18.2 for use in cancer diagnosis. Background technique [0003] Claudin is an integral membrane protein located within the tight junction of epithelium and endothelium. Claudin is predicted to have four transmembrane segments, with two extracellular loops, and both N- and C-termini located in the cytoplasm. The claudin (CLDN) family of transmembrane proteins plays a key role in the maintenance of epithelial-endothelial tight junctions and may also play a role in the maintenance of the cytoskeleton and in cell signal transduction. [0004] The claudin 18 (CLDN18) molecule is an integrated transmembrane prot...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/30C12N5/20G01N33/574C12R1/91
CPCC07K16/3046C07K2317/34C07K16/30A61P37/00C07K16/28C07K16/3023C07K16/303G01N33/57492G01N2800/52G01N33/574C12N5/163G01N33/531C07K2317/24
Inventor 乌尔·沙欣厄兹莱姆·图雷奇里塔·米特纳赫特-克劳斯斯特凡·韦尔
Owner ASTELLAS PHARMA INC
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