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Human tumour necrosin antibody, its preparation and medicinal composition

A technology of drugs and monoclonal antibodies, applied in the field of immunology, can solve the problems of low specificity, high immunogenicity, no diagnostic or therapeutic effect, etc., and achieve the effect of low price, advanced and simple preparation procedures

Inactive Publication Date: 2005-05-11
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, since most of the antibodies obtained in the above studies are mouse-derived antibodies, these antibodies have problems such as high immunogenicity, low specificity and / or insufficient stability to varying degrees, and there is no provision for a diagnostic or therapeutic function in vivo.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Construction of human single-chain antibody (scFv) gene library

[0032] (1). Preparation of cDNA

[0033] Collect 5ml of peripheral blood from 1000 people, mix them, and separate mononuclear cells with lymphocyte separation medium (produced by Tianjin Institute of Blood, Academy of Medical Sciences). Total mRNA was extracted from isolated human peripheral blood lymphocytes using a kit from Invitrogen. The mRNA purification kit from GIBCO was used to purify, and the mRNA obtained above was used as a template to reverse transcribe the first strand of cDNA. The above steps were carried out according to the instructions provided by the manufacturer.

[0034] (2).PCR amplification

[0035] Design and synthesize the following human antibody VH gene (hVH), VL gene (hVL), and 5′ end (back) with reference to the relatively conserved segment sequences of V region FR1 and FR4 in various human antibody gene family sequences that have been published at home and abroad , 3' end (f...

Embodiment 2

[0043] Cloning of ScFv fragments into phagemid vectors

[0044] After PCR was performed according to the above conditions, ScFv fragments with SfiI and NotI restriction sites at both ends were obtained, and purified by spin column chromatography to remove unnecessary adapter primers and dNTPs. The purified ScFv fragment was double-digested with SfiI and NotI (both purchased from Promega Company) to generate cohesive ends that can be connected to the vector pCANTAB5E (Pharmacia Company). Perform spin column chromatography purification again to remove small SfiI or NotI fragments that may affect its connection with the carrier.

[0045] The phagemid vector pCANTAB5E (Pharmacia Company) itself contains SfiI and NotI enzyme-cut cohesive ends, which can be connected with the above-mentioned ScFv fragments. Using conventional DNA ligase, the ScFv fragment is cloned into the corresponding site on the phagemid vector. The adjacent position on the pCANTAB5E vector is the g3p gene, an...

Embodiment 3

[0049] Panning Antibody Library

[0050] For the antibodies in the phage display library obtained in Example 2, antibodies with high affinity were selected by panning technology.

[0051] Coat the enzyme-linked plate with recombinant human TNF-α (rhTNF-α) antigen (purchased from Shenzhen Jingmei Company), block with BSA, incubate for 2 h, add 50 μl phage antibody library (about 10 12 CFU) and incubated for 2 h, washed once with TBST (Tris 50mmol / L, NaCl 150mmol / L, Tween-20 0.5%, BSA 1%, pH7.5) (5 times in the second round, and after the third round) 10 times, 5 min each time), recover phage with 50 μl of eluate, adjust pH to neutral with neutralization buffer, infect Ecoli TG1, and carry out the next round of screening. A total of 3 rounds of screening were performed to remove phagemids without antigen binding ability.

[0052] A sandwich ELISA experiment was performed to measure the antigen-binding activity of the antibody. Add 50 μl of 200ng / ml recombinant human TNF-α (rh...

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Abstract

This invention relates to a full human anti-TNF-alpha monocloned antibody with strong specificity and low immunogenicity as well as its preparation and medicinal composition therewith.

Description

technical field [0001] The present invention relates to the field of immunology; more specifically, the present invention relates to tumor necrosis factor antibody, its coding sequence, preparation method and application. Background technique [0002] Tumor Necrosis Factor α (Tumor Necrosis Factor α, TNF-α) is a multifunctional immune regulatory molecule in the body, it can bind to receptors on the cell membrane to play a role, often causing the death of target cells (its name comes from This) or attract local accumulation of immune effector cells. TNF-α is a soluble homotrimeric subunit with a molecular weight of 17KD (Smith, et al., J. Biol. Chem. 262:6951-6954, 1987). A transmembrane-bound precursor TNF-α was also found with a molecular weight of 26KD (Kriegler, et al., Cell 53:45-53, 1988). Mononuclear macrophages can secrete TNF-α and TNF-β after being stimulated by endotoxin and some other stimuli, and some other cells can also secrete TNF-α. [0003] TNF-α plays a ...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P19/02A61P29/00A61P31/18C07K16/18C12N15/12
Inventor 陈列平
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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