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Human anti-human CD20 monoclonal antibody molecule and application thereof

A fully human and antibody technology, applied in the fields of application, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, etc., can solve the problem of unsatisfactory therapeutic agents

Active Publication Date: 2013-01-16
广州市流式生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, while the evidence obtained so far proves that CD20 is an effective target for immunotherapy, it is also found that the existing murine, chimeric or even humanized or fully human antibodies are as high as 375 mg / day due to their HAMA or HACA reactions. m 2 The dose and frequent injections caused by too short injection intervals are still not ideal as a therapeutic agent

Method used

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  • Human anti-human CD20 monoclonal antibody molecule and application thereof
  • Human anti-human CD20 monoclonal antibody molecule and application thereof
  • Human anti-human CD20 monoclonal antibody molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1. Construction of a large-scale fully human antibody library

[0075] This example describes how to construct a very large-scale fully human natural antibody library in Fab format. The Fab antibody library of the present invention was constructed by using blood samples from more than 3,000 healthy adults from different regions and different ethnicities, referring to the following documents, and the detailed construction process is described after the bibliography.

[0076] 1. Dantas, BC, et al, 2005, Construction of a human Fab phage display library from antibody repertoires of osteosarcoma patients. Gene. Mo. Res, 4(2): 126-140.

[0077] 2. Hiroshi, T, et al, 1999, Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica, Clinical and Diagnostic Labor Immunol, May 1999, 383-387.

[0078] 3. Wu, BP, et al, 2001, Construction and selection of the natural immune Fab antibody phage display library from patients with co...

Embodiment 2

[0097] Example 2 Using human CD20 to screen the fully human antibody library

[0098] The self-prepared 2F2 (HuMax-CD20, GenMab) Fab was used as a positive control, and the recombinant human CD20 was used as an antigen to screen the above HuLib. The panning process is as follows:

[0099] Add 1 small portion of HuLib to a 25ml cell culture square bottle pre-coated with recombinant human CD20 protein, and incubate at 37°C for 1 hour. After washing 20 times with PBS containing 1% Tween-20, 1 ml of logarithmic phase TG1 cells were added, and cultured with shaking at 37° C. for 16 hours. Centrifuge at 12000rpm for 10 minutes, and transfer the supernatant to a new 50ml test tube. Take 500 μl of supernatant, and pan again according to the above method, for a total of 4 rounds. After the final round, the obtained bacterial suspension was diluted to 100,000 cells / ml and selected on 1.5% agar plates containing 0.1% ampicillin to obtain single spots. Inoculate ten 96-well deep-well ...

Embodiment 3

[0109] Example 3 Molecular Evolution

[0110] All CDRs of the 7F2 clone were analyzed by the method of Cunningham et al. (Cunningham and Wells, Science, 244:1081-1085, 1989), and residues sensitive to glycine substitution were identified, which were preferred mutation sites. The following table shows the preferred amino acid positions in the 7F2 clone, where the underlined ones are extremely sensitive to substitutions, and the dotted ones are relatively sensitive to substitutions, and they are all preferred mutation positions.

[0111]

[0112] The above-mentioned preferred sites are mutated by oligonucleotide-mediated random mutation (Kunkel method), and further improved antibody molecules for these sites can be obtained by panning. The brief process is: in order to introduce mutations at the above preferred sites, mutation primers were designed according to the Kunkel method, and a Fab secondary antibody library was constructed using pCOMb3M as a vector. Using the method...

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Abstract

The invention relates to an anti-human monoclonal antibody and a coding nucleic acid sequence thereof, and especially relates to an anti-human CD20 monoclonal antibody having a high affinity and a low dissociation rate. The preferable anti-human CD20 monoclonal antibody comprises a human Fc region, a framework region, and a human light-chain and / or heavy-chain variable region obtained through molecular evolution. The invention also discloses medicinal compositions composed of the human antibody, an application of the human antibody in human disease treatment, and a method for using the human antibody in the treatment.

Description

field of invention [0001] The present invention relates to anti-human CD20 monoclonal antibody. The invention is particularly concerned with high affinity, low off-rate anti-human CD20 mAbs. The invention also discloses the application of the drug composed of fully human antibodies in the treatment of human diseases. Background technique [0002] CD20 protein, also known as human B lymphocyte-restricted differentiation antigen or Bp35, is a non-glycosylated hydrophobic transmembrane phosphorylated protein with a molecular weight of 35kD, expressed on the surface of all mature B cells, encoded by the MS4A1 gene ( Valentine et al, 1989, J Biol Chem 264(19):11282-11287; and Einfield et al, 1988, EMBO J7(3):711-717). [0003] In humans, CD20 is expressed throughout all but the first and final stages of B cell development; it is present from pre-pre B cells to memory cells, but not in pro-B cells or plasma cells ( Bona C, et al, 1996, Textbook of Immunology. Martin Soohoo, 2nd...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N1/15C12N1/19C12N1/21C12N5/10A01K67/027A01K67/033A01H5/00A61K39/395A61P35/02
CPCA61K39/395A01K67/033C07K2317/92C07K2317/21C07K16/28C12N5/10A61K2039/505A01H5/00C07K2317/55A01K67/027C07K16/2887A61P35/00A61P35/02A61P37/00
Inventor 刘庆法
Owner 广州市流式生物科技有限公司
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