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Anti-human CD20 humanized antibody, preparation method and application thereof

A technology of humanized antibody and heavy chain, which is applied in the field of its preparation and antibody, can solve the problems of losing the affinity of the original antibody and reducing it

Active Publication Date: 2014-05-07
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

However, simple CDR transplantation often reduces or even loses the affinity of the original antibody. This is because the FR region not only provides the spatial conformation environment of the CDR, but sometimes directly participates in the mutual binding of antigens and antibodies. Change to murine residues to restore antibody activity

Method used

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  • Anti-human CD20 humanized antibody, preparation method and application thereof
  • Anti-human CD20 humanized antibody, preparation method and application thereof
  • Anti-human CD20 humanized antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of anti-human CD20 monoclonal antibody 8E4 - preparation of monoclonal antibody by cell fusion hybridoma

[0041] Raji cells highly expressing CD20 were used to immunize BALB / c mice (purchased from Shanghai Experimental Animal Center), so that the B lymphocytes in the spleen could produce anti-human CD20 antibodies, and the splenocytes and NS of the immunized mice were collected. -1 (BALB / c mouse myeloma cells) fusion, HAT selective culture, after culture, anti-human CD20 positive clones were screened, and then subclones were screened after cloning to ensure that the antibody was produced by a single cloned cell , and then the culture supernatant of single clone cells was collected and purified by Protein G column to obtain the monoclonal antibody 8E4 against human CD20.

Embodiment 2

[0042] Example 2 Construction of Chimeric Antibody c8E4

[0043] Cloning of variable region gene of anti-human CD20 monoclonal antibody 8E4

[0044] Extract 2 × 10 6 Total RNA of hybridoma cell 8E4 secreting anti-human CD20 mAb. Select the appropriate positions of the heavy chain and light chain constant regions of the antibody (IgG2a, κ) and design three gene-specific primers GSP1, GSP2, and GSP3 respectively. Among them, GSP1 is the farthest from the variable region gene and is used for reverse transcription reaction, and GSP2 is used for reverse transcription reaction. In the first round of PCR amplification, GSP3 was used for nested amplification. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., the sequence is as follows GSP1-H, 5'-AGC TGG GAAGGT GTG CAC ACC ACT-3'; GSP2-H, 5'-CAG AGT TCC AGG TCA AGGTCA-3'; GSP3-H , 5'-CTT GAC CAG GCA TCC TAG AGT-3'.GSP1-L, 5'-TTGCTG TCC TGA TCA GTC CAA CT-3'; GSP2-L, 5'-TGT CGT TCA CTG CCATCA ATC TT-3' GSP3-L...

Embodiment 3

[0050] Example 3 Construction of 8E4 humanized antibody

[0051] Homology Modeling of the Three-dimensional Structure of Murine 8E4 Monoclonal Antibody Variable Region (Fv)

[0052] The three-dimensional structure of the variable region of the 8E4 mouse monoclonal antibody was simulated by using the Insight II program package of Accelrys. First, the protein structure database (Protein Data Bank, PDB) was searched for the template proteins of the 8E4 heavy chain and light chain variable region proteins with BLAST program. The antibodies with the highest homology (PDBNO.2OSL) and (PDB NO.1WEJ) were selected as the modeling templates for the heavy chain and light chain of 8E4, respectively, with 84% and 94% homology, and were modeled using the Insight II program. The three-dimensional structure of 8E4, such as figure 1 shown.

[0053] Design and Construction of 8E4 Humanized Antibody

[0054] Human immunoglobulin heavy chain subgroup III (humIII) and Ig kappa chain subgroup I...

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Abstract

The invention belongs to the technical field of biology, and particularly discloses an anti-human CD20 humanized antibody hu8E4, a preparation method and application thereof. The amino acid sequences of heavy chain hyper-variable regions of the anti-human CD20 humanized antibody hu8E4 is CDR1: SYNMH, CDR2: AIYPENGDTSYNQKFKD and CDR3: WHYYGNGGALDY, and the amino acid sequences of light chain hyper-variable regions are CDR1: RASGNIHNYLA, CDR2: NAKTLPD and CDR3: QQFWSNPWT. The anti-human CD20 humanized antibody hu8E4 disclosed by the invention keeps the affinity and specificity of the original rat source antibody, has certain biological function, and can be used for preparing a lymphoma medicament for treating high-expression CD20.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to an antibody, its preparation method and application. Background technique [0002] Non-Hodgkin's lymphoma (NHL) is the most common malignant tumor of the lymphatic system, which occurs in young adults, and most of them are derived from B cells, accounting for about 85%. In the untreated state, the growth form of low-grade and some moderately malignant NHL such as small lymphocytic lymphoma and follicular lymphoma tends to be an indolent process, with a median survival period of 5 to 9 years. They are sensitive to the first chemotherapy, but are prone to relapse or drug resistance. When chemotherapy or radiotherapy is performed again, the curative effect is significantly reduced, so they are considered as difficult-to-cure malignant tumors. And some highly malignant NHL has a very high mortality rate. [0003] In recent years, antigen-targeted therapy targeting cell surface ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/85C12P21/08A61K39/395A61P35/00
Inventor 郭亚军李博华张大鹏李彩辉张彦
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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