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Whole human TNF alpha (tumor necrosis factor alpha) monoclonal antibody and preparation and application thereof

A monoclonal antibody and polynucleotide technology, applied in the fields of application, antibody, anti-inflammatory agent, etc., can solve the problem of low abundance of precursor cells, and achieve the effects of avoiding short half-life, reducing pain, and good affinity

Active Publication Date: 2011-07-20
优锐生物医药科技(深圳)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008]In addition, although the human immune system can produce an extremely large variety of antibodies, the technology of human or fully human antibodies has encountered a bottleneck: the precursor of specific antibodies produced in the human body Cell abundance is low, and even in antibody-positive individuals, the number of precursor cells capable of producing specific antibodies is far below the number of specific antibody-producing cells required for human hybridoma technology

Method used

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  • Whole human TNF alpha (tumor necrosis factor alpha) monoclonal antibody and preparation and application thereof

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Experimental program
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Effect test

Embodiment 1

[0024] The acquisition of embodiment 1 anti-human TNFa antibody gene

[0025] Take 5 milliliters of peripheral blood from patients with active rheumatoid arthritis, separate leukocytes with lymphocyte separation medium, culture them, and identify positive clones according to the results of ELISA.

[0026] 1. Select blood sample

[0027] In order to obtain human B cells that secrete anti-human TNFα, human TNFα (purchased from Shenzhen Jingmei Company) was used to routinely coat a 96-well plate, and each well was coated with 250 ng of the protein overnight. Then, it was blocked with 5% skimmed milk powder at room temperature for 2 hours, and the milk powder was prepared with pH7.2 PBS. After washing, 100 microliters of sera from different patients were added and incubated at room temperature for 1 hour. Then add peroxidase-labeled goat anti-human IgG and let stand at room temperature for 1 hour. After washing at least 5 times, add TMD or other chromogenic reagents and treat a...

Embodiment 2

[0050] The preparation of embodiment 2 anti-human TNFa antibodies

[0051] 1. Obtaining the heavy chain and light chain coding sequences

[0052] In order to express the gene of the full-length antibody, according to the sequencing results of Example 1, the full-length heavy chain and light chain sequences of human IgG1 were synthesized, so as to constitute a complete anti-human TNFa antibody-01 gene.

[0053] The following fragment is the light chain coding region, the length is: 645bp. (SEQ ID NO:1) GAAATTGTGCTCACACAGTCACCAGACTTTCAGTCTGTCACCCCTAAGGAGAAAGTGACCATCACTTGCAGGGCCTCTCAGTTCGTCGGCTATAGTATCC ACTGGTACCAGCAGAAACCCGATCAGTCCCCTAAACTGCTGATCAAGTACGCCTCTGAATCAAGGTCAGGTGTCCCCAGTCGATTTTCTGGATCAGGATC TGGTACCGACTTCACCCTCACCATCAATAGCTTGGAGGCCGAGGACGCTGCTACCTACTACTGCCAACAAAGCCACAGCTGGCACTTTACTTTTGGCCAG GGGACCAAGCTTGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT

[0054] 其编码的轻链氨基酸序列为(SEQ ID NO:3):EIVLTQSPDFQSVTPKEKVTITCRASQFVGYSIHWYQQKPDQSP...

Embodiment 3

[0070] PEGylation of embodiment 3 anti-human TNFa monoclonal antibody

[0071] The anti-human TNFa antibody obtained in Example 2 was structurally modified to introduce a free sulfhydryl group, and two protective agent amino acids AA were added after the 229th amino acid C to form a free sulfhydryl group, and further PEGylation, PEGylation and purification methods refer to APChampman et al. [Reference Literature: Therapeutic antibody fragments with prolonged in vivo halflife.Nature Biotech.V17 1999 August, Page 780-783] to obtain PEGylated anti-human TNFa monoclonal antibody. After identification by ELISA, it was confirmed that it reacted positively with TNFa. The prepared PEGylated antibody was tested for protection with L929 cells (ECACC 85011425) (refer to Example 5), and it was found that the PEGylated antibody had a good protective effect.

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Abstract

The invention relates to the technical field of biology, and discloses a whole human TNF alpha (tumor necrosis factor alpha) monoclonal antibody and preparation and application thereof. The invention adopts a whole human antibody technology and acquires an anti-human TNF alpha whole human antibody. Preliminary analyses on the physical, chemical and biological activities of the antibody indicate that the antibody has better affinity with TNF, and can effectively neutralize the killing effect of the TNF on L929 cells in vitro. The whole human TNF alpha monoclonal antibody disclosed by the invention minimizes the immunogenicity of a human body. A PEG (polyethylene glycol) surface modification technology is adopted, thus avoiding the defect of short half life of small molecule antibody, and the whole human TNF alpha monoclonal antibody is more suitable for application in vivo while keeping the anti-TNF alpha activity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a TNFα-binding protein and its application. Background technique [0002] Autoimmune diseases are the third largest group of diseases after cancer and cardiovascular diseases. Rheumatoid arthritis (RA) is one of the most common autoimmune diseases. The prevalence rate of rheumatoid arthritis in my country is about 0.32-0.36%, and the incidence rate among people over 60 years old is as high as 30%-40%. Faced with hundreds of thousands of yuan per person per year for imported drug treatment, patients often have to endure painful chemical treatment for a long time, but with little effect. [0003] RA has a large number of patients and a large amount of medication, which makes the market for therapeutic drugs huge. In 2005, the global market reached 6 billion US dollars. At present, there are many clinical treatment options for RA. The first choice is to use high-dose non-steroidal ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N15/13C12N1/19C12N5/10C12P21/08A61K39/395A61P29/00A61P19/04
CPCC07K16/241C07K2317/92C07K2317/21A61K2039/505A61P19/02A61P19/04A61P29/00A61P37/02
Inventor 倪健
Owner 优锐生物医药科技(深圳)有限公司
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