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

A technology of humanized antibodies and amino acids, applied in the biological field, can solve the problems of loss of affinity and reduction of the original antibody

Active Publication Date: 2011-05-11
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

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation of Anti-human CD20 Monoclonal Antibody 8F6 - Preparation of Monoclonal Antibody by Cell Fusion Hybridoma

[0042] 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 collect the culture supernatant of a single clone cell and purify it through a Protein G column to obtain the anti-human CD20 monoclonal antibody 8F6.

Embodiment 2

[0043] Example 2 Construction of Chimeric Antibody c8F6

[0044] Cloning of variable region gene of anti-human CD20 monoclonal antibody 8F6

[0045] Extract 2 × 10 6 Total RNA of hybridoma cell 8F6 secreting anti-human CD20 mAb. Select appropriate positions of antibody (IgG3, κ) heavy chain and light chain constant regions to design three gene-specific primers GSP1, GSP2, GSP3 respectively, among them, GSP1 is 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., and the sequences are as follows: GSP1-H, 5'-GTA GAG GTC AGA CTG CAG GAC-3'; GSP2-H, 5'-CTC AGG GAA ATA GCC CTT GAC-3'; GSP3 -H, 5'-AGA TCC AGG GGC CAG TGG ATA GAC-3'.GSP1-L, 5'-TTG CTG TCC TGA TCA GTC CAA CT-3'; GSP2-L, 5'-TGT CGT TCA CTG CCA TCA ATC TT-3'; GSP3-L, 5'-TTG TTC ...

Embodiment 3

[0052] Example 3 Construction of 8F6 humanized antibody

[0053] Homology Modeling of the Three-dimensional Structure of Murine 8F6 Monoclonal Antibody Variable Region (Fv)

[0054] The three-dimensional structure of the variable region of the 8F6 murine monoclonal antibody was simulated using the Insight II program package of Accelrys. First, the template proteins of the 8F6 heavy chain and light chain variable region proteins were searched in the protein structure database (Protein Data Bank, PDB) with BLAST program. The antibodies with the highest homology (PDB NO.2E27) and (PDB NO.1FH5) were selected as the modeling templates for the heavy chain and light chain of 8F6, respectively, with 84% and 94% homology, and were modeled using the Insight II program The three-dimensional structure of 8F6, such as figure 1 shown.

[0055] Design and Construction of 8F6 Humanized Antibody

[0056] Human immunoglobulin heavy chain subgroup III (humIII) and Ig kappa chain I subgroup (...

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Abstract

The invention belongs to the biotechnology field and in particular discloses an anti-human CD20 humanized antibody hu8F6 and a preparation method and application thereof. The amino acid sequences of the hypervariable region of the heavy chain of the anti-human CD20 humanized antibody hu8F6 are CDR1:NYWMQ, CDR2:AIYPGDGDTRYTQKFKG and CDR3:EGAYGYDDGMDY. The amino acid sequences of the hypervariable region of the light chain of the anti-human CD20 humanized antibody hu8F6 are CDR1:RASQSIRNNLH, CDR2:YASQSIS and CDR3:QQSNTWPLT. The anti-human CD20 humanized antibody hu8E4 disclosed by the invention retains the affinity and specificity of the original mouse antibody, has certain biological functions and can be used for preparing the drugs for treating lymphoma in a high CD20 expression state.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to an antibody, its preparation method and use. 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 molecule...

Claims

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Application Information

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Patent Type & Authority Applications(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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