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Anti human RANKL monoclonal antibodies developed by PAE technology and uses thereof

An antigen and antibody fragment technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, resistance to vector-borne diseases, etc. Safety, cost reduction effect

Inactive Publication Date: 2012-10-17
刘庆法
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] In the past, scientists were only able to create murine mAbs, which cannot be used in immunotherapy without humanization due to the HAMA response

Method used

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  • Anti human RANKL monoclonal antibodies developed by PAE technology and uses thereof
  • Anti human RANKL monoclonal antibodies developed by PAE technology and uses thereof
  • Anti human RANKL monoclonal antibodies developed by PAE technology and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Panning for variable region fragments specific to human RANKL

[0081] (1) All-human source combination Construction of antibody library

[0082] Using more than 3,000 blood samples from blood donors from different provinces and different nationalities, an ultra-large-scale antibody library was constructed with reference to the methods provided in the following literature. The phage lysate prepared from the constructed antibody library was diluted to 10E10 with YT medium containing 7% DMSO, then divided into 1 ml aliquots, and stored at -80°C for later use.

[0083] 1. Hoogenboom HR, and G Winter, 1992, By-passing immunisation: human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J MolBiol, 227(2): 381-388.

[0084] 2. Griffiths AD, SC Williams, O Hartley, IM Tomlinson, P Waterhouse, WL Crosby, RE Kontermann, PT Jones, NM Low, TJ Allison, TD Prospero, HR Hoogenboom, A Nissim, JPL Cox, JL Harrison, M Zaccolo, E Gherardi, G Wi...

Embodiment 2

[0117] Example 2: Preparation of recombinant monoclonal antibody protein

[0118] 1. Preparation of endotoxin-free plasmid DNA: inoculate 100ml LB medium with bacteria with phCMV-II / 3K7F5L and phCMV-II / 3K7F5H plasmids, and prepare plasmid DNA with Qiagen's Ultrapure Plasmid DNA Purification Kit.

[0119] 2. Transfection and culture of CHO cells: Transfect mammalian cells with the plasmid DNA prepared above and Invitrogen's LipoFamine2000. The transfection process is carried out by the method provided by the manufacturer. Alternatives such as PEI35000 can also be used.

[0120] 3. Transfection conditions: (1) Add CHO DHFR(-) cells (ATCC No. CRL-9096) to 5ml of newly prepared Ex302 medium (purchased from Sigma-Aldrich) to a final density of 2×10E5 cells / ml. After 48 hours incubation at 37°C, the cells were collected by centrifugation at 450×g for 10 minutes. (2) Add 1ml of fresh DMEM medium, tap the bottom of the tube gently to resuspend the cells. Then, the cells were transfected w...

Embodiment 3

[0123] Example 3: Expression of Fab format and preparation of recombinant protein

[0124] (1) Construction of expression vector

[0125] The DNA fragments carrying the aforementioned SEQ ID NO: 1 and SEQ ID NO: 3 were cloned into the pCOM3H vector (Wu SC, Lin YJ, Chou JW, Lin CW. 2004, Construction and characterization of a Fab recombinant protein for Japanese encephalitis virus neutralization. Vaccine. 25, 23(2):163-71). The 5'-light chain with NheI and NotI sites was digested with NheI / NotI and ligated to pCOM3H pre-cut with the same enzyme combination. Then the heavy chain with XhoI and SpeI is digested with the same enzyme combination and then ligated to its XhoI / SpeI site. After transforming the competent cells of Escherichia coli DH5α, the single clones were tested and isolated on an agar plate containing IPTG / X-gal and ampicillin, and the selected clones were confirmed by restriction digestion. A correct clone was inoculated into 500ml LB medium containing ampicillin and...

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Abstract

The invention discloses antibodies that bind high specificity to human RANKL (Receptor Activator for Nuclear Factor kappa B Ligand), wherein, the antibodies are used for the treatment of bone erosion caused by astogeny, hormonotherapy, postmenopausal osteoporosis, bone metastasis, and inflammation. The invention also discloses DNA sequences and supposed amino acid sequences of the antibodies.

Description

Technical field [0001] The present invention relates to a recombinant antibody that binds to human nuclear factor kappa B receptor activator ligand (Receptor Activator for Nuclear Factor kappa B Ligand, RANKL), and describes the effect of this type of antibody on the formation of osteoblasts as well as in autoimmune diseases and malignant tumors. And the potential use in the treatment of bone diseases such as osteoporosis and femoral erosion caused by hormone therapy. Background technique [0002] The formation and reconstruction of bone is a very complex biological process involving RANK, RANKL and OPG (osteoprotegerin). [0003] Osteoclasts and osteoblasts determine bone mass, bone structure and bone strength through their respective roles in bone resorption and bone formation. Bone remodeling is a spatially coordinated process that spans a lifetime, in which old bone is removed by osteoclasts and replaced by an osteogenic process with osteoblasts. In many pathological conditio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61P19/10A61P19/00
CPCC07K2317/21A61K2039/505C07K2317/76C07K16/2875C07K2317/74C07K2317/55A61P19/00A61P19/10Y02A50/30
Inventor 刘庆法
Owner 刘庆法
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