Antihuman CD52 monoclonal antibody hybridoma cell line, monoclonal antibody, engineered antibody, carrier, reagent kit and application thereof

A monoclonal antibody and antibody technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, fusion cell, etc. Damage and other problems, to achieve the effect of easy mass production, low antigenicity, and low production cost

Active Publication Date: 2013-03-20
UNION STEMCELL & GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While these drugs kill leukemia cells, they also seriously damage normal hematopoietic cells, so they have serious toxic and side effects on the body.
In addition, leukemia cells are often resistant to chemotherapy drugs, and the recurrence rate after chemotherapy is high, which seriously affects the effectiveness of clinical chemotherapy drugs

Method used

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  • Antihuman CD52 monoclonal antibody hybridoma cell line, monoclonal antibody, engineered antibody, carrier, reagent kit and application thereof
  • Antihuman CD52 monoclonal antibody hybridoma cell line, monoclonal antibody, engineered antibody, carrier, reagent kit and application thereof
  • Antihuman CD52 monoclonal antibody hybridoma cell line, monoclonal antibody, engineered antibody, carrier, reagent kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Preparation and fluorescent labeling of mouse anti-human CD52 monoclonal antibody

[0051] Using isolated human tonsil cells as antigens, immunize 6-week-old Balb / c mice with conventional methods, and use Freund's complete adjuvant for the first basic immunization, and perform basic immunization every 3 weeks, a total of three times, 2×10 7 Cells per mouse per time, booster immunization by intrasplenic injection 3 weeks after the last basic immunization, 1×10 7 Cells / mouse; 3 days later, the spleen was taken, fused with NS1 mouse myeloma cells, and carried out according to the conventional hybridoma preparation method. After 12 days of fusion, the supernatant of fusion wells grown by monoclonal growth was collected, and CD52-positive target cell lines were selected for screening and identification by indirect immunofluorescence method. Hybridoma cells with good reactivity and good growth status were selected for subcloning, and stable secretion of anti-human ...

Embodiment 2

[0054] Example 2 Anti-human CD52 monoclonal antibody detection of CD52 expression on the cell surface of leukemia cell lines

[0055] Take well-grown REH cells and collect them by centrifugation, and prepare 1×10 cells with PBS 7 / ml, each 100 μl was added to a flow tube, one tube was added with PE-labeled CD52 antibody, and the other tube was added with PE-labeled mouse IgG1 isotype control, and reacted at room temperature for 20 minutes in the dark. Add about 2ml of PBS to wash once, centrifuge at 1000rpm for 10 minutes, discard the supernatant, add 300μl of PBS, and detect by flow cytometry (see Figure 11 ).

Embodiment 3

[0056] Example 3 Anti-human CD52 monoclonal antibody can effectively inhibit the growth of leukemia cell lines in vitro

[0057] Take a well-growing CD52-positive leukemia cell line and adjust it to 1×10 with RPMI1640 culture medium containing 20% ​​FBS 5 / ml, add 100μl per well to a 96-well culture plate. Cells were divided into 4 groups. Control group 1: without adding any antibody; control group 2: adding 20 μg / ml goat anti-mouse secondary antibody; experimental group 1: adding 10 μg / ml Campath-1; experimental group 2: adding 10 μg / ml Campath-1 and 20 μg / ml ml goat anti-mouse secondary antibody. Place in a 37°C, 5% carbon dioxide incubator for 24-144 hours.

[0058] (1) Changes in cell morphology. After the leukemia cell line was co-cultured with HI186, it could obviously show a dispersed morphology, and no longer had the characteristics of aggregate growth. When HI186 acted alone or in combination with the second antibody, although the number of cells decreased to a c...

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PUM

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Abstract

The invention discloses an antihuman CD52 monoclonal antibody hybridoma cell line, a monoclonal antibody, an engineered antibody, a carrier, a reagent kit and application thereof, capable of specifically combining human CD52 antigen inside and outside a human body and purposefully removing normal immunological cells and killing alive leukemia cells through the single or coordinative role of various mechanisms. The invention relates to an antihuman CD52 monoclonal antibody HI186 heavy chain and light chain variable region gene, a polypeptide coded by the gene, a carrier containing the gene, and application of the gene and the polypeptide in preparing medicaments for diagnosing and treating leukemia or autoimmune diseases. The heavy chain and light chain variable region gene is from an antihuman CD52 monoclonal antibody. The invention successfully prepares a human single-chain antihuman CD52 gene engineering antibody by adopting a gene engineering technology and provides a novel effective medicament for diagnosing and treating the leukemia diseases and diseases of immune systems.

Description

technical field [0001] The invention relates to an anti-human CD52 monoclonal antibody hybridoma cell line, a monoclonal antibody, which can specifically recognize human CD52 molecules, and can be used for diagnosis and prognosis judgment of leukemia, for removing normal immune cells or for treating leukemia and autoimmune diseases. Engineered antibodies, vectors, kits and uses thereof. Background technique [0002] Leukemia is one of the main diseases that threaten human life and health. The incidence rate in my country is about 2.76 / 100000 people. Among them, acute lymphoblastic leukemia is more common in children, acute myeloid leukemia is more common in adults, and chronic leukemia is more common in people over 40 years old. It is a class of malignant diseases originating from hematopoietic (or lymphoid) stem cells. Due to stem cell damage, leukemia cells lose the ability to further differentiate and mature, or the proliferation and differentiation capabilities are unb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/12C07K16/28C12N15/13C12N15/63A61K39/395A61K48/00A61P35/00A61P35/02A61P37/06G01N33/577G01N33/574
Inventor 郭桂庆何大水黄丽华廖晓龙屈浩佘鸣王冬梅袁向飞张金香张丽艳张毅张宇光周立朱卫彬
Owner UNION STEMCELL & GENE ENG
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