Anti-tumor necrosis factor alpha human-derived antibody

A technology of humanized antibody and necrosis factor, which is applied in the direction of anti-tumor drugs, antibodies, anti-cytokine/lymphokine/interferon immunoglobulin, etc., and can solve problems such as attack, immunogenicity reduction, coma, etc.

Active Publication Date: 2012-09-19
LIVZON MABPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kind of chimeric antibody is to transplant the non-human antibody variable region to the human antibody constant region, and retain the original amino acid sequence of the heavy chain and light chain variable region of the non-human antibody (see, PCT of Daddona, P.E., etc. Publication No. WO92/16553, Le, U.S. Patent No. 5,919,452 of J. et al., PCT Publication No. WO2005/047329 such as Kang, Heui H, Chinese Patent Publication CN1544466A No. of Jin Boquan etc.), Chinese Patent Publication CN101177453 No. such as Jin Yihui provides a A new chimeric antibody that binds to human tumor necrosis factor. Although the immunogenicity of this chimeric antibody is reduced compared with non-human antibodies, the degree of murineization in the chimeric antibody is still high. This may lead to varying degrees of HAMA responses, specifically ma

Method used

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  • Anti-tumor necrosis factor alpha human-derived antibody
  • Anti-tumor necrosis factor alpha human-derived antibody
  • Anti-tumor necrosis factor alpha human-derived antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Example 1: Production of anti-hTNFα mouse monoclonal antibody

[0142] 1) immunity

[0143] Some 7- to 11-week-old female Balb / c mice were immunized IP or ID with recombinant hTNFα (rhTNFα, purchased from Pepro Tech Inc.), emulsified with an equal volume of TITERMAX or complete Freund’s adjuvant , with a final volume of 100-400 μL. Each mouse was IP (1-400 μg) and SC after 1-7, 5-12, 10-18, 17-25 and / or 21-34 days with an equal volume of TITERMAX or TNF emulsified in Freund's incomplete adjuvant. (1400 μg×2) for immunization. Mice can be bled after 12-25 and 25-40 days by post-frame puncture without anticoagulation. The blood was then allowed to coagulate for 1 hour at RT and the serum was collected and titered with TNFαELA according to known methods. Fusions were performed when repeated injections did not result in an increase in titer. At this point, mice can be given a final booster injection with 1-400 μg TNFα diluted in 100 μL saline. After 3 days, mice were ...

Embodiment 2

[0148] Example 2: Qualitative anti-hTNFα murine antibody

[0149] There are two assays for the characterization of anti-hTNFα antibodies. One method assays the antibody's competitive binding of adalimumab to hTNFα, and the other assays the antibody's ability to neutralize hTNFα in a L929 cytotoxicity assay. The two methods and their experimental results are described respectively below.

[0150] 1) Competition binding assay with adalimumab

[0151] Horseradish peroxidase (HRP)-labeled anti-hTNFα human antibody adalimumab was used as a reagent. RhTNF (50 μl, 0.05 g / mL) was coated on the microtiter plate and left overnight at room temperature. The coating solution was discarded, the wells were blocked with 1% skim milk dissolved in phosphate buffered saline (PBS) for 0.5 hours, and the wells were washed with PBS containing 0.05% Tween 20. Then a mixture of 50 μl of growth medium and 50 μl of HRP-labeled adalimumab was added to each well. Unlabeled adalimumab and medium with...

Embodiment 3

[0154] Example 3: Cloning of heavy and light chains of TM2-11-12 murine antibody

[0155] 1) Cloning of heavy chain variable region of TM2-11-12 murine antibody

[0156] In order to design the humanization of the murine antibody, it is first necessary to obtain the DNA fragments containing the coding sequences of the heavy chain and light chain variable regions of the murine antibody TM2-11-12. 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: 24) and 3' primer (5'-TCCAGGGGCCAGTGGATAGAGAGA-3', SEQ ID NO: 25) The heavy chain variable region DNA fragment, the 3' primer is homologous and antisense to the mouse IgG1 heavy chain constant region. These resulting DNA fragments were cloned into TOPO TA vector (Invitrogen) and sequenced. The amino acid sequence (SEQ ID...

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Abstract

The invention provides an anti-tumor necrosis factor human-derived monoclonal antibody and application of the human-derived monoclonal antibody. Compared with the traditional mouse chimeric antibody, the anti-tumor necrosis factor human-derived monoclonal antibody has the advantages that the antigen identifying capability of the antibody is kept, and the immunogenicity of mouse-derived antibody is greatly reduced, so that the clinical application security of the antibody is improved.

Description

technical field [0001] The present invention relates to at least one humanized antibody against tumor necrosis factor-α (TNFα) or its fragments, including specific parts or variants thereof, as well as nucleic acids encoding said humanized anti-TNFα antibodies, complementary nucleic acids, and vectors thereof , host cell and preparation method thereof, composition, kit and application thereof comprising said anti-TNFα humanized antibody. Background technique [0002] Human tumor necrosis factor-α (TNFα) is a pro-inflammatory cytokine produced by monocytes and macrophages. It is initially produced as a 26kDa precursor protein with the N-terminal in the cell and the C-terminal in the extracellular. Known as transmembrane TNFα, Pennica et al first cloned the TNF-α gene cDNA in 1984, and deduced that the human TNF-α molecule is composed of 157 amino acids, and its molecular weight is about 17KD (Pennica D, et al, Nature 1984; 312 :724). Human TNFα has two molecular forms, name...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/13C12N15/63C12N5/10G01N33/577A61K39/395A61P31/04A61P37/02A61P35/00A61P11/00A61P37/06A61P31/18A61P31/22A61P29/00A61P33/06
CPCC07K16/241C07K2317/24A61K2039/505A61P11/00A61P25/00A61P29/00A61P31/04A61P31/18A61P31/20A61P31/22A61P33/06A61P35/00A61P37/02A61P37/06A61P43/00C07K2317/33C07K2317/76C07K2317/90C07K2317/92Y02A50/30
Inventor 朱保国陶德胜孙乃超金宜慧刘瑞贤周若芸李强彭育才张经纬胡振湘
Owner LIVZON MABPHARM
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