Recombined chimeric antibody against human tumor necrosis factor alpha

A tumor necrosis factor, chimeric antibody technology, applied in the field of immunology, can solve problems such as attack

Active Publication Date: 2008-05-14
PHARMAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, these foreign antibodies may be attacked by host immune antib...

Method used

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  • Recombined chimeric antibody against human tumor necrosis factor alpha
  • Recombined chimeric antibody against human tumor necrosis factor alpha
  • Recombined chimeric antibody against human tumor necrosis factor alpha

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Production of anti-hTNFα mouse monoclonal antibody

[0083] Recombinant hTNFα (rhTNFα, purchased from PeproTech Inc.) 20 μg was injected subcutaneously into four-week-old BALB / c mice in complete adjuvant. It is injected every three to four weeks for a total of five times. Finally, a single injection of 20 μg rhTNFα was intraperitoneally. After serum testing, mice with high levels of anti-hTNFα antibody serum were identified. The mouse spleen was taken out and fused with the mouse myeloma Sp2 / 0 cell line. Mixed 5×10 8 Sp2 / 0 cells and 5×10 8 Spleen cells are fused in 50% polyethylene glycol (PEG, molecular weight 1450) and 5% dimethylsulfoxide (DMSO) solution. Use Iscove medium (containing 10% fetal bovine serum, 100 units / ml penicillin, 100μg / ml streptomycin, 0.1mM hypoxanthine, 0.4μM aminopterin and 16μg thymidine) to adjust the number of spleen cells to 7.5×10 5 / ml, add 0.2ml to the wells of 96-well culture plate. Place at 37°C with 5% CO 2 Inside the incubator....

Embodiment 2

[0084] Example 2 Characterization of anti-hTNFα murine antibody:

[0085] There are two methods for qualitative anti-hTNFα antibody. One method is to measure the antibody's competitive binding to Humira and hTNFα, and the other method is to measure the ability of the antibody to neutralize hTNFα in the L929 cytotoxicity assay. The two methods and their experimental results are described below.

[0086] 1. Competitive binding assay with Humira antibody

[0087] Horseradish peroxidase (HRP, Boehringer Manheim) labeled anti-hTNFα human antibody Humira (Abbott) was used as a reagent. RhTNFα (50 μl, 0.05 μg / ml) was coated on an ELISA plate (CorningLife Sciences) at room temperature overnight. The coating solution was discarded, and each well was blocked with 1% skimmed milk dissolved in phosphate buffered saline (PBS) for 0.5 hours, and the wells were washed with PBS containing 0.05% Tween 20. Then add 50 μl growth medium (DMEM+5% FBS, Invitrogen) and 50 μl HRP-labeled Humira antibody ...

Embodiment 3

[0090] Example 3 Cloning the heavy and light chains of TM2-11-12 murine antibody

[0091] In order to express the C2-11-12 chimeric antibody, DNA fragments encoding the variable regions of the heavy and light chains of the anti-hTNFα murine antibody TM2-11-12 must be obtained first. RNA was isolated from TM2-11-12 mouse hybridoma cells using an RNA purification kit (Invitrogen Corp.) to prepare cDNA (GeneRacer kit, Invitrogen Corp.). Isolation from cDNA by polymerase chain reaction (PCR) using 5'primer (5'-CGACTGGAGCACGAGGACACTGA-3', SEQ ID NO: 11) and 3'primer (5'-TCCAGGGGCCAGTGGATAGACAGA-3', SEQ ID NO: 12) Heavy chain variable region DNA fragment. The 3'primer is homologous and antisense to the constant region of the mouse igG1 heavy chain. The DNA fragments obtained were cloned into TOPO TA vector (Invitrogen) and sequenced. All heavy chain variable region clones showed the same nucleotide sequence (SEQ ID NO: 1). This nucleotide sequence and its encoded amino acid sequence (SE...

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Abstract

Described herein are antibodies that bind to human tumor necrosis factor alpha (hTNFα). And list the nucleotide sequence of antibody heavy chain and light chain variable region and its derived amino acid sequence. The heavy chain and light chain variable region genes are respectively connected with the human immunoglobulin gamma 1 (hIgG1) heavy chain constant region and human kappa (k) light chain constant region genes to form a chimeric gene. Then, the vector containing the chimeric gene is introduced into the host cell line to express the antibody protein. This recombinant chimeric antibody protein can neutralize the activity of hTNFα in vitro, and is suitable for treating patients with excessive secretion of harmful hTNFα, including certain inflammations, such as rheumatoid arthritis and Crohn's disease.

Description

Technical field [0001] The present invention relates to the field of immunology, in particular to chimeric antibodies and their uses, in particular to recombinant chimeric antibodies against human tumor necrosis factor alpha and their uses. Background technique [0002] Human tumor necrosis factor alpha (hTNFα) is a cytokine produced by many cell types, including macrophages, mast cells, and lymphocytes. The biological activity of human TNFα conducts signal transduction through two different receptors (p55 and p75), which have important regulatory effects on inflammation and the immune system. In various reports, it is believed that hTNFα is involved in the pathophysiology of many diseases, including sepsis, autoimmune diseases, transplant rejection, infectious diseases and intestinal dysfunction (see, for example, Vasilli, P., Annu. Rev. Immunol. 10 :411-452, 1992; Tracey, K., and Cerami, A., Annu. Rev. Med. 45:491-503, 1994). Since hTNFα is harmful in many patients, antagonists...

Claims

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Application Information

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IPC IPC(8): C07K14/525C07K19/00A61K38/19A61P35/00C12N15/13C12N15/63
Inventor 金宜慧孙乃超周若芸刘瑞贤
Owner PHARMAB
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