Monoclonal antibody for anti-human CEA, a composition containing same and application thereof

A technology of antibody and antibody light chain, applied in the field of molecular biology and biology

Active Publication Date: 2009-12-23
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although no antibody targeting CEA has been approved by the FDA so far, a large number of anti-CEA antibody drugs are in clinical and preclinical research

Method used

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  • Monoclonal antibody for anti-human CEA, a composition containing same and application thereof
  • Monoclonal antibody for anti-human CEA, a composition containing same and application thereof
  • Monoclonal antibody for anti-human CEA, a composition containing same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Preparation and identification of monoclonal antibody CC4.

[0066] Application of hybridoma technology (Kohler and Milstein 1975; Yeh, Hellstrom et al.1979; Yeh, Hellstrom et al.1982) produced and screened to obtain antibody CC4. The brief description is as follows: BALB / c mice (Beijing Experimental Animal Center) were immunized with living human colorectal cancer cell LS 174T cells (ATCC) as antigen, and the mice were killed after 4 booster immunizations, and the spleen was taken and removed. Cells were suspended in RPMI medium. Splenocytes were fused with SP2 / 0-Ag14 murine myeloma cells (ATCC) in the presence of polyethylene glycol (PEG), and hybridomas were selected using HAT-selective medium. See reference (Kohler and Milstein 1975) for specific methods.

[0067] Using fixed LS 174T cells cultured in a 96-well cell culture plate as an antigen, the culture supernatants of hybridoma cells were screened by ELISA. The brief introduction of the cell ELISA ...

Embodiment 2

[0077] Example 2: Study on the specificity of monoclonal antibody CC4 to tumor tissue and tumor cells.

[0078] In order to systematically study the binding specificity of the monoclonal antibody CC4 of the present invention to human normal and tumor tissues, 29 cases of human normal tissues and 24 cases of frozen sections of human tumor tissues were screened by immunohistochemical method.

[0079] The specific experimental method is as follows: human normal tissue or tumor tissue was embedded with OCT (frozen tissue embedding fluid, provided by Sakura), and frozen section was performed. Sections were fixed in pre-cooled acetone for 5 min, then in 0.3% HO 2 o 2 Incubate in dark methanol at room temperature for 30 minutes to remove the interference of endogenous peroxidase. After washing with PBS for three times, the sections were blocked with 5% horse serum (Beijing Zhongshan Jinqiao, horse serum diluted in PBS), and then in the primary antibody dilution solution (1:2000 blo...

Embodiment 3

[0097] Example 3: Antigen identification of monoclonal antibody CC4.

[0098] Using western blotting, it was found that CC4 recognized a 130kD protein present in LS 174T cells and colorectal cancer tissues. The specific operation is as follows: collect the human-derived colorectal cancer cell LS 174T (ATCC) that strongly binds to CC4, wash the cells twice with pre-cooled PBS, centrifuge at 800 rpm at 4°C for 5 minutes, and use lysate (Tris-HCl 50mM pH 8.0, NaCl 150mM, EDTA 1mM, NP-40 1%, Glycerol 10%, PMSF 100μg / ml) to lyse the cells, centrifuge at 12000g at 4°C for 15 minutes, collect the supernatant, add DTT (final concentration 100mM) (dithiothreose Alcohol) and DTT-free loading buffer (5x loading buffer: 0.313M Tris-HCl, pH6.8, 10% SDS, 0.05% bromophenol blue, 50% glycerol), cook at 100°C. Whole-cell proteins were separated by 10% SDS-PAGE, followed by semi-dry electrotransfer to nitrocellulose membranes. After blocking with 5% skimmed milk, CC4 ascites was used as the p...

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Abstract

The invention relates to a mouse monoclonal antibody of anti-human carcino-embryonic antigen (CEA) resisting colorectal neoplasm activity, prepared by a biological technology. The method comprise a method for detecting CEA family protein by utilizing the antigen-combining capacity specificity of the mouse monoclonal antibody CC4 of anti-human CEA and a neoplasm treatment method by utilizing the colorectal neoplasm activity. The antibody can be used for specifically recognizing the human CEA at molecular, cellular and texture levels and recognizing a special antigen epiposition widely distributed in the CEA family protein. The CC4 is specifically combined with colorectal neoplasm texture at the texture level and presents stronger capabilities of restraining neoplasm growth, neoplasm metastasis, neoplasm invasion in an animal model and at the cellular level. The antibody and detection and treatment methods based on the antibody can become an effective neoplasm diagnosis and treatment tool in basal research or clinical application.

Description

technical field [0001] The invention belongs to the fields of molecular biology and biotechnology. Specifically, the present invention relates to an anti-human CEA mouse monoclonal antibody CC4, which can specifically bind colorectal cancer tissue and effectively inhibit the growth, migration and invasion of colorectal tumor cells at the animal model and cell level. CC4 can specifically recognize human CEA protein at the biochemical, cellular and tissue levels, and bind to an antigenic epitope conserved in CEA family proteins. Utilizing these characteristics, the antibody can be widely used in the clinical diagnosis and treatment of tumors expressing CEA or CEA family proteins. Background technique [0002] Carcinoembryonic antigen (CEA) is one of the earliest tumor-associated antigens discovered by people. It was first discovered and reported in colon cancer tissue extracts by Gold P and Freedman SO in 1965 (Gold and Freedman 1965; Gold and Freedman1965). In 1966, Gold P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K16/30C12N5/20C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/395A61K49/00A61P35/00G01N33/68
Inventor 阎锡蕴冯静郑超固杨东玲卢迪
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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